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Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachochytrium dendrobatidis
Laia Ribas,
Affiliation Department of Infectious Disease Epidemiology, Imperial College London, United Kingdom
Ming-Shi Li,
Affiliation Department of Paediatrics, Imperial College London, London, United Kingdom
Benjamin J. Doddington,
Jacques Robert,
Affiliation University of Rochester Medical Centre, Rochester, New York, United States of America
Judith A. Seidel,
J. Simon Kroll,
Lyle B. Zimmerman,
Affiliation Developmental Biology, National Institute for Medical Research, London, United Kingdom
Nicholas C. Grassly,
Trenton W. J. Garner,
Affiliation Institute of Zoology, Regent's Park, London, United Kingdom
Matthew C. Fisher
* E-mail: matthew.fisher@imperial.ac.uk
Laia Ribas Ming-Shi Li ... Matthew C. Fisher
Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.
Citation: Ribas L, Li M-S, Doddington BJ, Robert J, Seidel JA, Kroll JS, et al. (2009) Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachochytrium dendrobatidis. PLoS ONE 4(12): e8408. https://doi.org/10.1371/journal.pone.0008408
Editor: Gustavo Goldman, Universidade de Sao Paulo, Brazil
Received: September 18, 2009; Accepted: November 20, 2009; Published: December 22, 2009
Copyright: © 2009 Ribas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by United Kingdom Biotechnology and Biological Research Council (BBSRC) grant BB/E0232071 and United Kingdom Natural Environmental Research Council (NERC) grant NE/E006701/1 to MCF and TWJG, a Royal Society grant to MCF, a George John and Sheilah Livanos Charitable Trust grant to MSL, and National Institutes of Health (NIH) grant R24-AI-059830 to JR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd) [1], [2], [3], [4]. While anthropogenic spread of Bd appears to be driving the global distribution of the pathogen [5], disease and declines are modulated by a complex interaction between environment, host and pathogen-specific factors [6], [7]. Research has identified temperature as a key factor in predicting the spatial occurrence of Bd-related declines [8], [9], and longer-term climate trends appear to drive these patterns of disease by amplifying the growth of Bd at its thermal optimum [6], [7]. However, this model ignores the potential contribution of host immunity. While it is known that amphibian antimicrobial peptides (AMPs) correlate with species survival against Bd [10], [11], it is not known if amphibians mount a specific response against Bd, or to what extent any host response is temperature-dependent. Amphibians are ectotherms, and seasonal temperature variability exerts strong direct effects on their immune system [12]. As a consequence, the susceptibility of amphibians to pathogens is expected to vary between seasons. Across longer temporal scales, global warming is negatively affecting survival of even widespread habitat-generalists such as the common toad Bufo bufo [13]. Therefore, the outcome of infection by Bd is likely to be exacerbated by synergies between short-term temperature-dependent effects on the immunocompetence of animals and longer-term physiological stressors.
Here, we describe the use of mathematical models and post-genomic technologies to identify the pathogen-associated gene expression profiles that a model amphibian species, Silurana (formerly Xenopus) tropicalis, exhibits in response to infection by Bd at different temperatures. We focus on defining the temporal dynamics of infection at two infection time-points, days 7 and 42, and at two temperatures; these temperatures were chosen to reflect the 'tropical' environmental norm for S. tropicalis, 26°C, and a lower, non-optimal 'temperate' temperature, 18°C. By using these two temperatures, we shifted the animals' environment towards the growth optimum for Bd (in vitro between 17°C and 25°C (Figure 1) [14]. In order to quantify the importance of host immunity and Bd growth-rate at these two different temperatures, we profiled the prevalence and intensity of Bd burdens in frogs over a 42-day infectious time course. By using a simple mathematical model, we estimated the contributions of host-response and Bd-growth to the observed infectious outcomes. In tandem, we investigated variation in the host response by profiling time and temperature-dependent global gene expression profiles in the main lymphopoietic tissues in frogs, the spleen by using microarrays and qRT-PCR transcriptomic strategies. Finally, we compared our data with the recent publication of the S. tropicalis transcriptome-response to Bd exposure at cool (18°C) temperatures [15].
Figure 1. Experimental infection and model outputs.
A. Observed prevalence data for iCT animals (blue circles) and modelled prevalence data (blue line, RSS (residual sum of squares) = 0.112), and observed prevalence data for iWT animals (red circles) and modelled prevalence data (solid red line, RSS = 1.99). Dotted orange line shows modelled iWT prevalence data where an identical host response to iCT animals has been assumed. g means the gap between modelled prevalence in iWT with and without differing host response due to temperature. Modelling prevalence without taking into account a differing host response does not fit the observed data well (RSS = 1611). B. Third order polynomial fitted to measurements of temperature against daily exponential growth rate of Bd in culture from [14] (y = -0.00008639x3 + 0.002612x2 – 0.001124x – 0.0004419). Blue circle indicates Bd growth rate at the temperature of iCT animals, and red circle indicates Bd growth rate at the temperature of iWT animals. C. The intensity of infection measured by qPCR as zoospore genomic equivalents.
Bd Infection Dynamics
Quantitative Bd-specific PCR shows that the prevalence and intensity of infection peaked in animals at each temperature between days 7 and 26 (Figure 1A & C). This suggested that the frogs were mounting an anti-Bd response in both groups, as animals were exposed to high infections (∼106 zoospores per procedure) throughout the course of the experiment; in the absence of a host response levels of infection should climb throughout the lifetime of the experiment. There was a higher prevalence and intensity of infection in iCT (infected Cold Temperature group) compared to iWT (infected Warm Temperature group) frogs throughout the experiment (Figure 1). Fitting a mathematical model of the infection process (Figure 1A) showed that animals in the iWT treatment had approximately a third of the chance of infection per procedure compared with those in the iCT treatment (βiWT = 0.170 and βiCT = 0.451) and, once infected, iWT animals recovered almost five times as fast (γiWT = 0.071 and γiCT = 0.015). After accounting for the greater Bd growth rate at the cooler temperatures, the fit of our model was consistent with iWT frogs clearing zoospores approximately twice as fast as iCT frogs (c iWT = 1.322 and c iCT = 0.646). Modelling prevalence assuming an identical host response in iWT animals as in iCT animals does not fit the observed data well (Figure 1). This finding suggests that iWT frogs mount a more effective immune response to Bd than do iCT frogs.
Overview of Patterns of Differential Gene-Expression
To identify the molecular basis of the host response to Bd infection, we used microarrays to profile gene expression in the cold and warm infected cohorts at days 7 and 42 of infection (see Figure S1 for the experimental design). We focused on the spleen which, in the absence of lymph nodes, is the major lymphoid organ in S. tropicalis. This profiling revealed that there was a robust infection-specific aspect to the global induction of gene expression following exposure to Bd, and this response was both temperature- and time-dependent. From a total of 10,878 probes, following normalization to the controls, 434 genes for the iCT and 492 genes for the iWT groups were differentially regulated throughout the experiment. On day 7, frogs had received two exposures to Bd with the result that the ratio of significantly up-regulated:down-regulated genes (the "up∶down ratio") in iCT frogs was 133∶95, compared to a ratio in iWT frogs of 96∶88. By day 42, after twelve successive exposures to Bd, iCT frogs showed an up∶down ratio of 105∶81 compared to 71∶121 for iWT frogs. Within these differentially expressed groups of genes, iWT frogs expressed significantly (Student t-test: p<0.01) higher numbers of genes with a fold change >30 (2.3%) and 2.5–1.5 (74%) relative to iCT frogs where fold change ranged between >30 (1.83%) and 2.5–1.5 (56.5%). There was little overlap in expression profiles between these two time-points within groups, even when the stringency of the statistical analysis was reduced to p<0.05; here iCT frogs shared 3.30% of genes between days 7 and 42 and iWT frogs shared 2.62%. This finding shows that both infected groups are changing their expression profiles during the intervening 35 day period to a large degree. Temperature-independent genes that were shared between iCT and iWT groups represented 4.76% of the total expression profiles. Differentially regulated genes (>1.5 fold and p<0.01) in Bd infected groups compared against controls are summarized in Table S1. Principal component analysis (PCA) of gene expression in the spleen (Figure 2B) showed that early (day 7) iCT frogs contributed the most diverse patterns of gene-expression of any group, as well as showing the highest prevalence and intensities of infection (Figure 1). These animals were in 'double jeopardy' because they faced both a non-optimal temperature [16], [17] and infection by Bd that is growing close to, or at, its environmental optimum [14], [18].
Figure 2. Microarray results in spleen of S. tropicalis Bd infected.
A. Expression of the top 50 genes obtained from microarrays with known or putative immune-function as shown by two-way gene clustering and clustering analysis (GeneSpring Software). The colour scale shows the fold changes above normalised controls. B. Principal Component Analysis (PCA) showing that S. tropicalis frogs held at cold temperatures (iCT) have the most diverse transcriptional profile during the first stage (7 days) of infection. Axis: X = PCA Component 1 (24.99% variance); Y = PCA Component 2 (18.12% variance); Z = PCA Component 3 (14.95% variance).
Differentially-Expressed Genes in 'Warm Infected' Frogs
Strikingly, the most highly expressed gene in the spleen of iWT frogs 7 days after Bd exposure was the AMP preprocaerulein type III precursor (PPCP; Figure 2, 3). Microarray analysis showed that this gene was found to be expressed 59 fold change above controls in spleen (Table S1, Figure 3A). The decapeptide caerulein is a principal constituent of the skin secretion of a variety of amphibian species [19], [20], [21] and is the most potent AMP to be secreted by S. tropicalis, with a demonstrated antimicrobial activity against 22 microbial species including Gram-positive bacteria and the fungus Candida albicans [22]. The strong up regulation of the differential PPCP expression observed by the microarrays was further validated by qRT-PCR individually for the spleens of all 52 experimental frogs (WT and CT groups; Figure 3B). Interestingly, expression of PPCP was not detected in spleen, or at low levels, in the control group, and there was no significant induction of this gene in any of the CT groups. qRT-PCR showed that PPCP expression levels were up to 71-fold upregulated in day 7 iWT frogs, confirming our microarray results. These results suggest that PPCP is specifically upregulated by S. tropicalis in response to Bd infection, and that induction only occurred at temperatures that are optimal for the host. Production of PPCP-protein has not previously been observed from tissues other than granular skin glands [23], however further analysis using qRT-PCR of skin-tissue showed no clear evidence of PPCP RNA production (Ribas, unpub. obs.). Furthermore, the recent publication by Rosenblum et al. [15] of the transcriptomic response in the liver and skin of infected S. tropicalis by Bd at 18°C also did not find any expression of AMPs in either of these tissues. Together, these findings raise the interesting and potentially important hypothesis that the spleen could be involved in manufacturing and the subsequent trafficking of this peptide following Bd infection as a first response of the innate system, and that this only occurs early on in infection, and at host-optimal temperatures. However, further work is needed to investigate this hypothesis, including an investigation of the temporal nature of AMP secretion and it's efficacy in the skin against Bd, following infection.
Figure 3. Microarray validation by quantitative RT-PCR for each of the 52 experimental frogs using spleen RNA.
For qRT-PCR analysis, samples were analysed in triplicates for each individual (control and infected). A. Microarray validation by qRT-PCR amplification for a subset of 10 genes with known or suspected roles in immunity. Black bars = microarray data (pooled samples); White bars = qRT-PCR validation data (individual samples). Data is expressed by log2 Relative Quantification versus the control group.. B. Further transcriptomic analysis by qRT-PCR of the expression of PPCP and trypsinogen 2 genes individually tested for all frogs (n = 52). Data is expressed by log2 Relative Quantification versus the control group (iWT7/cWT7, iWT42/cWT42, iCT7/cCT7 and iCT42/cCT42) ± Standard Error. * Indicates significance by Student t-test (p<0.05) compared to the control group at each time point.
Within the iWT cohort, levels of expression of PPCP had reduced to background by day 42 (Figure 3B) showing a strong temporal dependency in the expression of this gene. The production of AMPs is induced following exposure to various pathogens in other amphibian species, such as Rana esculenta [24], [25]. Pathogen-specific induction of AMPs has been demonstrated for this species, where it was shown that it only synthesises AMPs when microbes (bacteria or fungi) are present [25]. The reduction in PPCP production over time that we observe is possibly correlated with the recurrent nature of our infection regime; frogs were challenged every 4 days with over 106 zoospores and such high levels of infectious assault are likely to exhaust the capacity of these frogs to synthesise, traffic and discharge, PPCP [26]. In addition to PPCP, a second AMP, the Cytastin C precursor peptide, was found to be upregulated by 2.6 fold (Table S1, Figure 2A, 3a) increasing the evidence of the key role of AMPs in protection of amphibians from pathogens [27]. However, as for the PPCP results described above, it is unclear whether increased expression of this protein in the spleen represents de novo synthesis of this AMP, or rather its presence in the spleen is a consequence of the system-wide trafficking of this precursor peptide.
It is estimated that X. tropicalis genome has around 28,000 genes, therefore the Operon v1.0 S. tropicalis microarray that we use here represents a subset of around 40% of these genes and lacks probes for many of the immune genes involved in the anti-pathogen response. Nevertheless, cluster analysis identified a number of genes with homology to vertebrate genes with known roles in immunity. We identified 50 genes with known immune-associated function, and demonstrate the upregulation of several other genes with suspected roles in vertebrate innate immunity (Figure 2A). Notably, genes encoding cell-surface receptors were upregulated over the first stages of infection, indicating that pathways involved in innate-pathogen recognition are rapidly activated (Figure 2A, 3A). For example, tumour necrosis factor associated factor (TRAF, 1.5 fold) which interacts directly or indirectly with members of the TNFR superfamily [28], fucose binding lectin (2.3 fold) and leptin receptor (2.1 fold) were rapidly induced after two exposures of Bd. Subsequently, a down regulation of these pathogen-recognition genes were observed at the end of experiment (fucose binding lectin, -36.7 fold). Activation of other inflammatory markers, guanylate binding protein interferon inducible (1,44 fold), calcineurin interleukin (IL) 2 inducible (1,33 fold) and trypsinogen genes, were also observed (Figure 2, Table S1). Expression of these trypsinogen genes were significantly increased after 42 days of exposure, although this effect was not as strong as in animals infected at cold temperatures (Figure 2 and 3B, see below). Genes responsible for hemopoietic stem cell maintenance such as VENTX2 (2.6 fold) and hematopoietic SH2 protein adaptor (1.5 to 26 fold) were also increased at 7 days of infection, suggesting that hematopoietic cell development in the spleen is upregulated as a result of infection [29].
In marked contrast to the upregulation of genes involved in innate immunity, the expression of several genes with roles in the generation of an adaptive immune response were downregulated in the spleen over the experiment. Such genes include the B-cell translocation gene (−1.88 fold, day 7) and the cutaneous T-cell lymphoma associated antigen (−1.69 fold, day 42). The complement factor B, an element of the complement system which bridges the innate and adaptive immunity with a critical importance for the generation of a potent humoral response [30] was also inhibited throughout the infection in iWT frogs (−1.63 fold, day 7 and −3.33 fold, day 42; Table S1, Figure 2A). Our results are in concordance with the findings of Rosenblum et al. [15] where five complement pathway genes were shown to decrease their expression in S. tropicalis liver after Bd infection [15]. In this study, these authors raise the hypothesis that a lack of evidence for an adaptive immune response to Bd both in liver and skin tissues is suggestive of a more complex, perhaps inhibitive, effect of Bd on the immune system. Although more needs to be done to understand the role of specific-immunity in combating Bd infection, our experimental results suggest a central role of the amphibian spleen as an effector of the innate immune system, and support the conclusion that there is little evidence for an active adaptive immune-reaction.
Differentially-Expressed Genes in 'Cold Infected' Frogs
Microarray data showed that expression of the AMP PPCP did not occur in iCT frogs (Figure 2A). In order to confirm the lack of the expression of this skin peptide in this group, we individually analysed the expression of PPCP by qRT-PCR. Expression of this gene amongst individual iCT frogs was significantly lower than that found among the iWT group (Figure 3B), validating the microarray findings (Figure 2A). Principal component analysis of gene expression in the spleen (Figure 2B) showed that in particular, the iCT 7 day group had the most divergent transcriptional pattern; this was in concordance with the mathematical model where frogs subjected to non-optimal temperatures presented the highest prevalence and intensities of infection. Among the top 50 highly up-regulated genes in the spleen of Bd infection in the cold group, seven genes corresponded to serine protease transcripts (trypsinogens, chymotrypsin, zymogen and elastases; Figure 2A). Serine protease proteins are involved in several photolytic pathways but are also recognised effectors of the innate immune system and are associated with generalised inflammation [31], [32]. Our analysis showed a large increase in levels of expression from day 7 to day 42 for these genes (19 fold change; Figure 3B) in iCT animals and a similar, but less pronounced effect, was seen in the iWT frogs (Figure 3B). Here, we speculate that iCT frogs are attempting to compensate for their lack of PPCP-production by upregulating the production of serine proteases as a secondary, inflammatory, innate immune response. The production of serine proteases are pro-inflammatory markers as they represent an increase in mast-cell activity and degranulation [33], [34]; this may well provide a protective role as our prevalence data and modelling show that even the iCT frogs are able to control their Bd infections, albeit to a lesser degree than is seen in the iWT groups.
In mammals, inflammatory immune reactions are catalysed by the cross-linking of mast-cells with IgE, and this constitutes the major host-response to mucosal gastrointestinal nematodes [35]. The inverse relationship seen here between PPCP and protease production in animals subjected to cold temperatures suggests that a negative feedback may occur between these two facets of the S. tropicalis innate immune response, i.e. that inflammatory responses and secreted AMPs may be mutually inhibitory. If this is the case, then the reduction of AMP-activity seen in iCT and late-stage iWT animals may be the major factor allowing an upregulation of these inflammatory cell-responses.
Wider Conclusions
We have infected the African Pipid frog S. tropicalis at two temperatures and profiled the group prevalence and intensities of infection over 42 days. This has shown that both frogs held at 'optimal' temperatures (iWT; 26°C) and those held at 'non-optimal' temperatures (iCT; 18°C) are able to survive very high, repeated, doses of Bd. Profiling infection using qPCR has shown that both groups of frogs manifest an early peak of infection followed by a reduction in prevalence; this suggests that both groups of frogs are clearing infection however that the frogs held at 'optimal' temperatures clear their infections faster. We then asked the question 'does this effect occur due to either the increased growth of Bd at lower temperatures or to more effective host-responses at warmer temperatures?' We use a simple mathematical model parameterised on measured Bd growth rates at different temperatures to address this question. Our findings show that, while the Bd growth-rates can explain some of the difference between the prevalence of infection between iCT and iWT groups of frogs, the rate of zoospore clearance needs to be far higher for the iWT animals in order to explain our data, even when taking into account the decrease in Bd growth-rates at 26°C (shown as the differences between the 'blue' and 'red' spots in Figure 1B). This difference in zoospore clearance between the iWT and iCT frogs is shown as the difference g in Figure 1A. As the rate of zoospore clearance is analogous to the effectivity of the host response, these findings suggest that frogs held at 'optimal' 26°C temperatures are mounting a response that is able to clear Bd infections.
We then identified the basis of this host response in 'optimal' iWT compared to 'non-optimal' iCT frogs by profiling the changes in the host transcriptome for early (day 7) and late (day 42) infections in the major amphibian lymphoid tissue, the spleen. Because we were interested in identifying ecologically-relevant effects, we chose to house animals in groups rather than singly. While this prevents our analysis from looking at individual-level effects (our transcriptional-level responses are for the experimental groups pooled between tanks), our results are ecologically relevant as S. tropicalis are colonial in nature and cohabit ponds. Further, a pooling strategy enabled us to extract enough rRNA from these frogs small spleens for direct Cy3/Cy5 labelling; this enabled us to avoid pre-amplifying the RNA using PCR, a process which introduces unknown biases in gene-expression. Recent studies by Kendzidorski et al. [36] have shown that most genes in expression studies are not adversely affected by pooling strategies and that the increase in sample-sizes (52 in this study) outweigh the loss of power in ascertaining individual-level-responses.
Our analyses of the S. tropicalis spleen-transcriptome showed extensive differences between all of our combinations of experimental groups. Because the frogs spleens were pooled between tanks, we removed tank-level effects. Therefore, the residual changes in gene-expression are attributable to our three major variables, 'infection', 'time' and 'temperature'; all of which contribute to major changes in expression levels in the S. tropicalis spleen. Despite the complex patterns that we found, we were able to isolate three major signals that discriminated between infected and uninfected animals. These were (1) strong upregulation of innate immune AMP-effectors at host optimal temperatures early in infection, (2) ablation of this response in frogs housed at 18°C and (3) a serine-protease proinflammatory response in both optimal and non-optimally-housed frogs.
One of the great conundrums surrounding the emergence of Bd has been its extraordinarily wide host-range in over 350 species, the greatest seen for any vertebrate pathogen to date ([37]; www.spatialepidemiology.net/bd-maps). It has long been suspected that this wide distribution may result from the ability of the pathogen to manipulate and subvert host-immunity or to avoid detection. Recent data by Rosenblum et al. [15] have provided important experimental evidence for this hypothesis by showing that several genes that are associated with immune functions, such as toll-like pathogen receptors and complement-pathway genes, are downregulated in infected S. tropicalis. Our data also support this hypothesis by showing that putative markers of B-cell, T-cell and complement factors are downregulated in the spleens of animals that have been infected at host-optimal 26°C temperatures. Unfortunately, a full comparison between ours, and Rosenblum et al.'s studies is not possible due to the limited gene-set (40%) that are represented on the Operon AROS microarrays that we have used, preventing effective cross-comparisons. However, recent unpublished data targeting a wider panel of adaptive immune-markers in the S. tropicalis liver by qPCR confirm our general findings by showing that these markers are all downregulated in infected frogs at host-optimal temperatures (Seidel, Ribas & Fisher, unpub. data). Taken together, these observations stemming from independent studies by different research groups are broadly concordant in their conclusion that there is a generalised down regulation in markers of adaptive immunity in S. tropicalis following infection by Bd.
Work on another class of eukaryotic pathogens exhibiting wide host ranges, the helminths, has shown that these parasites exhibit widespread subversion of host immunity; strategies include skewing the host response towards a tolerogenic TH2 responses, interference with antigen-processing and modulation of antigen-presenting cells [35]. Interestingly, the final two processes are thought to be modulated by nematode homologues of mammalian cystatins, which function to maintain mammalian dendritic cells in their immature state in order to downregulate antigen presentation [38]. In this study we found that the cystastin C precursor peptide was upregulated in iWT frogs when Bd burdens were highest (at day 7), suggesting that a modulation of host immunity by this mechanism may be occurring. Searching the Bd genome for close homologues of vertebrate proteins with known roles in immune evasion and down-regulation of host immune responses should provide a fruitful approach to identifying whether these specific virulence factors occur. If found, such virulence factors would provide evidence that a long co-evolutionary history has occurred between Bd and its Pipid hosts [39], [40]; this is predicted by the 'Out of Africa' hypothesis advocated by Weldon et al. accounting for the global spread of this pathogen owing to the international trade in Pipid species [41].
It is not clear from our study why markers of adaptive immunity are downregulated in iWT frogs. However, down regulation of S. tropicalis' adaptive immunity may be a consequence of the route of infection (cutaneous) and the primary immunity (AMP-mediated and inflammatory innate immunity). The clearest finding from our study was that the most over-expressed transcript (up to x70 fold) was associated with an innate-effector AMP. The various arms of the vertebrate immune response are interdependent, and the upregulation of one arm has extensive effects on the expression of other immune modes; the TH1/TH2 inhibitory dicotomy is a well known example of this effect. It may be that over-expression of AMPs has a dampening effect on other limbs of the amphibian immune-system, resulting in under-expression of adaptive-immune effectors. However, it is perhaps naïve to expect that the effects that we are describing here can be captured by simple 'innate versus adaptive' models, and a systematic network analysis of S. tropicalis interactomes on an individual basis is required to dissect these interactions [42]. Recently reported preliminary results demonstrating the development of high-titer antibodies to Bd in Xenopus laevis injected with the heat-killed pathogen and subsequently exposed to live zoospores [43], suggest that Pipids are able to make anti-Bd antibodies. In this case, then the route of infection is likely to be important in determining what sort of host response is manifested. Importantly, our data show that, owing to the lack of a strong adaptive immune response but high innate response, it is unlikely that S. tropicalis can be primed to respond to subsequent challenges of Bd by using a cutaneous method of infection; we suggest that it will not be easy to develop vaccines against Bd and it is unlikely that wild populations will acquire a natural immunity to the pathogen.
Mathematical modelling of the dynamics of infection in our experimental treatments has shown that frogs maintained below their temperature optimum reduce the generation of a successful anti-Bd response, resulting in higher infectious burdens. Analysis of the iCT versus control CT and iWT animals suggests that this may be due to a lack of AMP production; there was no expression of PPCP in the iCT frogs. This may explain why Rosenblum et al. found no evidence of AMP upregulation in their whole-genome arrays as, in their experimental design, all frogs were housed at 18°C; these conditions correspond to our iCT group which was not found to express markers of AMP activity. These findings are likely to be ecologically relevant as it has been shown that, in the field, immunity is temperature-dependent and forced by seasonal patterns. For example seasonal surveys of adult Red spotted newts (Notophthalmus viridescens) by Raffel et al [12] showed a spring lag effect in lymphocyte levels and strong seasonal acclimatization of lymphocyte, neutrophil and eosinophil levels in the autumn. A body of work now exists on the prerequisite of a successful AMP-response to manifest resistance to chytridiomycosis at the species level [18], [44], [45]. Taken together, these studies suggest that factors that influence AMP production are likely to change whether populations/species are refractory to chytridiomycosis, or whether they succumb. It is important to note that there is an apparent association between declines of species and altitude, with populations at higher altitudes suffering greater losses and extinction rates [6], [46]. There is now a clear need for such field-based immunological studies among infected populations to investigate the importance of the host response in the epidemiological triad (host, Bd and the environment). Such studies will quantify the influence that changing climates are able to exert on the ectothermic host response to this devastating emerging pathogen.
All experiments involving amphibians were conducted under Licence from the British Home Office following the Animals (Scientific Procedures) Act of 1986. Welfare of the amphibians followed the principles of the 3R's; reduction, refinement and replacement and were overseen by a named Veterinarian as well as Animal Care and Welfare Officers. All experiments were preceded by full ethical review by the Imperial College Ethical Review Committee and the British Home Office, and were fully approved by these two organisations.
Silurana tropicalis Infection and Temperature Model
52 adult female frogs of the species S. tropicalis (average weight 12.6 g) were reared in 8 water tanks (3–4 frogs per tank) in temperature-controlled rooms stabilised at 18°C, cold temperature group (CT) or 26°C warm temperature group (WT); Figure S1. Frogs were infected with Bd by individual exposures to 106 Bd zoospores in a 100 ml bath for 3 hours (these groups are designated iCT or iWT). Bd strain IA042 passage 10 was isolated from infected Alytes obstetricans in the Pyrenees, Spain and cultured in ThGL liquid media; zoospore inocula were standardised by counting motile zoospores using a haemocytometer. Isolate IA042 is known to have high virulence from previous experiments [39]. Exposures were repeated twice a week for 7 or 42 days (iCT7, iCT42, iWT7, iWT42). Control animals were similarly treated with regular transfer to an experimental bath, but were exposed to ThGL medium as a sham-infection (groups CT7, CT42, WT7, WT42). Animal welfare was checked daily while feeding and equal amounts of food given to all groups. We observed no sign of illness beyond skin-sloughing early on in the experimental regime, and no mortality occurred. Throughout the experiment, dermal swabs were taken (days 0, 7, 26 and 42) from each animal and infection monitored by a Bd-specific TaqMan Assay (Boyle et al. 2004) (Figure S1). In order to ensure that we were working with a Bd–free colony, frogs were swabbed and tested prior to starting the experiment and were all found to be negative. At days 7 and 42, animals were euthanized by ten-minute exposure to an overdose of MS-222 before the spleen from each animal was rapidly dissected on ice and placed into RNAlater (Qiagen), then kept at −80°C until further transcriptomic analysis (microarray & qRT-PCR).
Bd Dynamics and Mathematic Modelling
The prevalence and intensities of infection were estimated using a qPCR Taqman assay [47]. Briefly, Bd DNA was extracted from swabs and a TaqMan-probe assay used in an Applied Biosystems 7300 Sequence Detection System. Samples were prepared in duplicate with negative controls, and a standard curve was used to quantify the number of Bd zoospore genomic equivalents. The observed prevalence of infection are shown in Figure 1A.
In order to dissect the relative importance of host immunity versus Bd growth-rates, a mathematical model of the experiment was created assuming a constant rate of clearance of infection, and infection only at infection procedures. The model splits the experimental animals into three categories: Susceptible (S), Infected (I) and Recovered (R). All animals begin in the Susceptible category, from which they move into the Infected category at βS animals per infection procedure (which occurred bi-weekly). Infected animals then recover into the Recovered category at rate γI (such that the time spent infected is exponentially distributed.). The equations for the model are given below. (1) (2) (3)
where β is the rate of infection per procedure, γ is the recovery rate once infected, and * indicates a change at a single time point.
This model makes two major assumptions: that the chance of infection via transmission from an infected animal is negligible compared to the chance of being infected by a procedure; and that once an animal recovers it now has either, or a combination of, such a low chance of infection that it will now no longer be infected, or such a fast rate of recovery that if infected it will clear zoospores fast enough that it will show up as negative when tested for infection – i.e. once recovered, animals will stay in the Recovered category. Transmission is assumed to be negligible compared to infection by procedure as the highest intensity of infection reported from any animal was 15.12 zoospore genomic equivalents, whereas each procedure exposed the animals to 106 zoospores, an amount several magnitudes greater than any possible exposure from other animals. To test whether reinfection can be assumed to be negligible, a model was created in which animals in the Recovered compartment were allowed to become reinfected, with different infection and recovery rates to that of newly infected animals. Fitting this model to the prevalence data gave a greatly reduced rate of infection and increased rate of recovery for re-infected recovered animals, supporting the assumption.
The average infected lifespan (T) for both iCT (infected 18°C) and iWT (infected 26°C) animals was calculated from the infection model as the inverse of γ. In order to discern whether differences in infected lifespan between the two models can be explained entirely by differences in Bd growth-rate, or whether a differing host response at each temperature was required, the host response necessary at each temperature in order to return the zoospore count to 0.1 genome-equivalents (the amount above which infection is indicated) at the end of the average infected lifespan was then calculated. This was done by assuming Bd growth within the host is exponential, with a rate determined by two opposing factors: the intrinsic exponential growth of Bd at that temperature (r), and the rate of zoospore clearing by the host (c). To determine r the daily exponential growth rate of Bd in culture was calculated for each temperature [48] and then fitted to a third order polynomial equation (Figure 1B) from which r was calculated for both experimental temperatures. As so little is known about the immune response of S. tropicalis to fungal infections, the simplest form possible for the rate of clearing zoospores has been assumed, with c remaining constant and acting from the moment of infection. Provided that the exponential growth rate of Bd in culture is comparable to the uninhibited growth rate of Bd on the host, and that further infection procedures have no effect on the number of zoospores infecting the host, the required rate of clearing can then be calculated using the equation Ze(r−c)T = 0.1, where Z is the number of zoospores initially infecting the host, and has been assumed to be directly proportional to the rate of infection and the number of zoospores used per procedure. The values of c for both warm and cold experiments were then compared. Mathematical models were fitted to infection dynamics using Berkeley Madonna 8.3.14.
Microarray Hybridization and Transcriptomic Analysis
RNA samples from all spleen tissues were individually prepared (n = 52) using Qiagen RNeasy Mini Kit Columns (Qiagen) and further treated with DNaseI (Invitrogen). RNA quality and quantity was assessed using Bioanalyzer 2100 with RNA 6000 Nano LabChip Kit (Agilent Technologies) and NanoDrop 1000 Spectrophotometer (NanoDrop Technologies). To achieve enough RNA for direct Cy5 labelling without resorting to PCR pre-amplification, RNA spleens from 2 or 3 animals were pooled to give 3 pools of spleen RNA samples for each group of animals (see Figure S1) following published studies [36]. Samples were pooled between tanks but within experimental groups to remove tank-level effects. Sample (4 µg) of each RNA-pool were labelled using SuperScript III cDNA synthesis kit, oligo d(T) primer (Invitrogen) and Cy5-dCTP (GE Healthcare/Amersham). The resulting products were purified using a QIAquick cleaning kit (Qiagen). In parallel, a universal reference RNA sample was created from the spleen of an outgroup of animals (n = 4), which was further amplified to achieve large quantities using Amino Allyl MessageAmp™ II aRNA Amplification (Ambion). The resulting aRNA was labelled using a Cy3 mono-Reactive Dye Pack (GE Healthcare/Amersham). This Cy3-labelled aRNA was served as an universal reference (see below) to provide consistent slide-to-slide and experiment-to-experiment comparability. The principle and reliability of universal reference system using pooled RNA or amplified RNA were originally described [49], [50].
A total of 24 Xenopus tropicalis V1.0 AROS DNA microarray slides were obtained from Operon Biotechnologies (www.operon.com) and previously described [51]. The array design is available at ArrayExpress (www.ebi.ac.uk) under accession number A-MEXP-1446. Slide hybridization and washing steps were performed according to manufacture's protocol using buffers provided with the kit (Operon Biotechnologies). Each array slide was co-hybridized with one of the Cy5 labelled experimental samples and Cy3 labelled aRNA (universal reference). The resulting slides were scanned using a GenePix 4000B (Axon) microarray scanner and the scanned images were analysed with GenePix Pro6.0 (Axon). Statistical analysis was carried out using Genespring GX 7.3 (Agilent). Background-subtracted spot intensities resulting from hybridisation of Cy5-labeled cDNA derived from experimental RNA (signal channel) were divided by the intensity of the signal derived from Cy3-labeled aRNA (control channel). Log-transformed ratios were normalized by applying the intensity-dependent data analysis technique LOWESS, using 20% of the data for smoothing. Only genes not marked absent by GenePix Pro analysis were included. If the value for the reference channel was less than 10 then a value of 10 was used instead. Data arising from the three pooled biological replicates were combined as follows:
For each gene, the normalized log-transformed ratio arising from samples harvested from the infection group kept at 26°C for 7 days (iWT7) was re-normalized to the median value, as was that arising from uninfected group kept at 26°C for 7 days (cWT7). These were combined to give a ratio iWT7/cWT7. Likewise, the ratio were also generated for iWT42/cWT42, iCT7/cCT7 and iCT42/cCT42, representing experimental groups conditioned at 26°C for 7 days, at 18°C for 7 days and 18°C for 42 days, respectively. Differentially expressed genes were identified as genes showing more than 1.5-fold up- or down-regulation (p≤0.01) using a Student t-test in GeneSpring. Fully annotated microarray data have been deposited under ArrayExpress accession number E-MEXP-1936. Expression values are shown in Table S1.
Microarray Validation
Microarray-based gene expression patterns were confirmed for a subset of 10 genes (Figure 3A, Table S2) with known or suspected roles in immunity, by using qRT-PCR. In order to maximise the power of our data, individual analyses were run for each of the experimental frogs. A total of 52 individual spleen RNA samples, 12 for control (CT, WT) and 14 for infected animals (iCT, iWT) for each treatment group (7 and 42 days), were reverse-transcribed using SuperScript III and oligo d(T) (Invitrogen). The resulting cDNA were used as templates in qRT-PCR run in Applied Biosystem 7100 as following: Cycle 1; 50°C 2 min., Cycle 2; 95°C 10 min., Cycle 3; 95°C 15 sec., 60°C 1 min for 40 times. Samples were run as triplicates, with water and RNA as negative controls. All expression values were normalised against the constitutively-expressed gene, elongation factor α. Relative gene expression levels for these qRT-PCR results were analysed using the 2−ΔΔCT method [52] showing log2 Relative Quantification versus the control (iWT7/cWT7, iWT42/cWT42, iCT7/cCT7 and iCT42/cCT42) ± Standard Error.
Further gene expression analysis was performed in order to assess the reliability of the transcriptomics results, for two target genes of interest; the skin peptide PPCP and the serine-protease trypsinogen 2. These two genes were individually tested by qRT-PCR from the spleen RNA all 52 experimental frogs prior to pooling. Statistical analyses of the Relative Quantification results were completed using the Student t-test in the R-Software Package.
The subset of validated genes were (Table S2): muscle specific gene (Xt_10001800), trypsinogen 2 (Xt_10000537), T-cell (Xt_10005209), CD82 antigen (Xt_10002930), TRAF (Xt_10007227), Preprocaerulein type III precursor (Xt_10003079), epithelial membrane protein 2 (Xt_10006495), cystatin C precursor (Xt_10001311), BCL2/adenovirus (Xt_10007462) and protein EST EC2BBA27CH07 (Xt_10007462).
Schematic of the experimental design. Frogs were infected with Bd by individual exposures to 106 Bd zoospores in a 100 ml bath for 3 hours (iCT or iWT). These exposures were repeated twice a week for 7 or 42 days (iCT7, iCT42, iWT7, iWT42). Control animals were similarly treated with regular transfer to an experimental bath, but exposed to ThGL medium as a sham-infection (CT7, CT42, WT7, WT42). Throughout the experiment, dermal swabs were collected from each animal and infection monitored by Bd-specific TaqMan Assay (*). For transcriptomic analysis RNA was extracted individually from spleen tissues. To achieve enough RNA samples for direct Cy5 labelling without amplification for microarray hybridization, spleen RNA from 2 or 3 animals were pooled to give 3 pools of spleen RNA samples for each animal group. In parallel, a universal reference RNA sample was created from spleen of an out-group animals (n = 4), and was largely amplified aRNA. 3 and 4 microarray slides were used for each experimental group for control and infected treatment respectively (a total of 24 microarray slides). Microarray validation and other transcriptomic analysis were performed by qRT-PCR individually for each animal group of the experiment.
Expression values of the microarray results in spleen of animals Bd infected after 7 or 42 days. For statistical analysis, see the Materials and Methods section of the manuscript. Fully annotated microarray data have been deposited under ArrayExpress with accession number E-MEXP-1936.
(0.18 MB XLS)
Primer information for the genes used in validating the microarray analysis.
(0.05 MB PDF)
We thank Professor Charles R. Bangham for comments on experimental design and the manuscript.
Conceived and designed the experiments: LR MCF. Performed the experiments: LR JAS TWJG. Analyzed the data: LR MSL BJD JAS NCG TWJG MCF. Contributed reagents/materials/analysis tools: LR MSL JR JSK LBZ TWJG. Wrote the paper: LR MSL BJD JR NCG TWJG MCF.
1. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues AS, et al. (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306: 1783–1786.
2. Berger L, Speare R, Daszak P, Green DE, Cunningham AA, et al. (1998) Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc Natl Acad Sci U S A 95: 9031–9036.
3. Gewin V (2008) Riders of a modern-day Ark. PLoS Biol 6: e24.
4. Rovito SM, Parra-Olea G, Vasquez-Almazan CR, Papenfuss TJ, Wake DB (2009) Dramatic declines in neotropical salamander populations are an important part of the global amphibian crisis. Proc Natl Acad Sci U S A 106: 3231–3236.
5. Fisher MC, Garner TW (2007) The relationship between the emergence of Batrachochytrium dendrobatidis, the international trade in amphibians and introduced amphibian species. Fungal Biology Reviews 21: 2–9.
6. Pounds JA, Bustamante MR, Coloma LA, Consuegra JA, Fogden MP, et al. (2006) Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439: 161–167.
7. Bosch J, Carrascal LM, Duran L, Walker S, Fisher MC (2007) Climate change and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; is there a link? Proc Biol Sci 274: 253–260.
8. Kriger KM, Hero JM (2007) Large-scale seasonal variation in the prevalence and severity of chytridiomycosis. Journal of Zoology 271: 352–359.
9. Muths E, Pilliodb DS, Livoc LJ (2008) Distribution and environmental limitations of an amphibian pathogen in the Rocky Mountains, USA. Biological Conservation 141: 1484–1492.
10. Rollins-Smith LA, Conlon JM (2005) Antimicrobial peptide defenses against chytridiomycosis, an emerging infectious disease of amphibian populations. Dev Comp Immunol 29: 589–598.
11. Woodhams DC, Ardipradja K, Alford RA, Marantelli G, Reinert LK, et al. (2007) Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses Animal Conservation 10: 409–417.
12. Raffel TR, Rohr JR, Kiesecker JM, Hudson PJ (2006) Negative effects of changing temperature on amphibian immunity under field conditions. Functional Ecology 20: 819–828.
13. Reading CJ (2007) Linking global warming to amphibian declines through its effects on female body condition and survivorship. Oecologia 151: 125–131.
14. Piotrowski JS, Annis SL, Longcore JE (2004) Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96: 9–15.
15. Rosenblum EB, Poorten TJ, Settles M, Murdoch GK, Robert J, et al. (2009) Genome-wide transcriptional response of Silurana (Xenopus) tropicalis to infection with the deadly chytrid fungus. PLoS One 4: e6494.
16. Cooper EL, Wright RK, Klempau AE, Smith CT (1992) Hibernation alters the frog's immune system. Cryobiology 29: 616–631.
17. Maniero GD, Carey C (1997) Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold. Journal of Comparative Physiology B-Biochemical Systemic and Environmental Physiology 167: 256–263.
18. Woodhams DC, Alford RA, Briggs CJ, Johnson M, Rollins-Smith L (2008) Life-history trade-offs influence disease in changing climates: strategies of an amphibian pathogen. Ecology 89: 1627–1639.
19. Erspamer V, Erspamer GF, Mazzanti G, Endean R (1984) Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea. Comp Biochem Physiol C 77: 99–108.
20. Erspamer V (1984) Half a century of comparative research on biogenic amines and active peptides in amphibian skin and molluscan tissues. Comp Biochem Physiol C 79: 1–7.
21. Brevins CL, Zasloff MA (1990) Peptides from frog skin. Annu Rev Biochem 59: 395–414.
22. Ali MF, Soto A, Knoop FC, Conlon JM (2001) Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). Biochim Biophys Acta 1550: 81–89.
23. Seki T, Kikuyama S, Yanaihara N (1989) Development of Xenopus laevis skin glands producing 5-hydroxytryptamine and caerulein. Cell Tissue Res 258: 483–489.
24. Simmaco M, Mangoni ML, Boman A, Barra D, Boman HG (1998) Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora. Scand J Immunol 48: 357–363.
25. Mangoni ML, Miele R, Renda TG, Barra D, Simmaco M (2001) The synthesis of antimicrobial peptides in the skin of Rana esculenta is stimulated by microorganisms. FASEB J 15: 1431–1432.
26. Tyler MJ, Stone DJM, Bowie JH (1992) A Novel Method for the Release and Collection of Dermal, Glandular Secretions from the Skin of Frogs. Journal of Pharmacological and Toxicological Methods 28: 199–200.
27. Rollins-Smith LA (2009) The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines. Biochimica et Biophysica Acta (BBA) - Biomembranes 1788: 1593–1599.
28. Arron JR, Walsh MC, Choi Y (2002) TRAF-mediated TNFR-family signaling. Curr Protoc Immunol Chapter 11: Unit 11 19D.
29. Moretti PA, Davidson AJ, Baker E, Lilley B, Zon LI, et al. (2001) Molecular cloning of a human Vent-like homeobox gene. Genomics 76: 21–29.
30. Fujita T, Endo Y, Nonaka M (2004) Primitive complement system–recognition and activation. Mol Immunol 41: 103–111.
31. Owen CA, Campbell EJ (1999) The cell biology of leukocyte-mediated proteolysis. J Leukoc Biol 65: 137–150.
32. Campbell EJ, Cury JD, Shapiro SD, Goldberg GI, Welgus HG (1991) Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases. J Immunol 146: 1286–1293.
33. Pejler G, Abrink M, Ringvall M, Wernersson S (2007) Mast cell proteases. Advances in Immunology, Vol 95 95: 167–255.
34. Rauter I, Krauth MT, Westritschnig K, Horak F, Flicker S, et al. (2008) Mast cell-derived proteases control allergic inflammation through cleavage of IgE. Journal of Allergy and Clinical Immunology 121: 197–202.
35. Maizels RM, Yazdanbakhsh M (2003) Immune regulation by helminth parasites: Cellular and molecular mechanisms. Nature Reviews Immunology 3: 733–744.
36. Kendziorski C, Irizarry RA, Chen KS, Haag JD, Gould MN (2005) On the utility of pooling biological samples in microarray experiments. Proc Natl Acad Sci U S A 102: 4252–4257.
37. Fisher MC, Garner TWJ, Walker J (2009) The global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time and host. Annual Review of Microbiology 63: 291–310.
38. Pierre P, Mellman I (1998) Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93: 1135–1145.
39. Fisher MC, Bosch J, Yin Z, Stead DA, Walker J, et al. (2009) Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence. Mol Ecol 18: 415–429.
40. Rosenblum EB, Stajich JE, Maddox N, Eisen MB (2008) Global gene expression profiles for life stages of the deadly amphibian pathogen Batrachochytrium dendrobatidis. Proc Natl Acad Sci U S A 105: 17034–17039.
41. Weldon C, du Preez LH, Hyatt AD, Muller R, Speare R (2004) Origin of the amphibian chytrid fungus. Emerging Infectious Diseases 10: 2100–2105.
42. Calvano SE, Xiao WZ, Richards DR, Felciano RM, Baker HV, et al. (2005) A network-based analysis of systemic inflammation in humans. Nature 437: 1032–1037.
43. Rollins-Smith LA, Ramsey JP, Reinert LK, Woodhams DC, Livo LJ, et al. (2009) Immune defenses of Xenopus laevis against Batrachochytrium dendrobatidis. Front Biosci (Schol Ed) 1: 68–91.
44. Rollins-Smith LA, Woodhams DC, Reinert LK, Vredenburg VT, Briggs CJ, et al. (2006) Antimicrobial peptide defenses of the mountain yellow-legged frog (Rana muscosa). Developmental and Comparative Immunology 30: 831–842.
45. Woodhams DC, Rollins-Smith LA, Carey C, Reinert L, Tyler MJ, et al. (2006) Population trends associated with skin peptide defences against chytridiomycosis in Australian frogs. Oecologia 146: 531–540.
46. Bosch J, Martínez-Solano I, García-París M (2001) Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain. Biological Conservation 97: 331–337.
47. Boyle DG, Boyle DB, Olsen V, Morgan JA, Hyatt AD (2004) Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat Organ 60: 141–148.
48. Piotrowski JS, Annis SL, Longcore JF (2004) Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96: 9–15.
49. Novoradovskaya N, Whitfield ML, Basehore LS, Novoradovsky A, Pesich R, et al. (2004) Universal Reference RNA as a standard for microarray experiments. BMC Genomics 5: 20.
50. Park PJ, Cao YA, Lee SY, Kim JW, Chang MS, et al. (2004) Current issues for DNA microarrays: platform comparison, double linear amplification, and universal RNA reference. J Biotechnol 112: 225–245.
51. Graindorge A, Thuret R, Pollet N, Osborne HB, Audic Y (2006) Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray. Nucleic Acids Res 34: 986–995.
52. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408.
Is the Subject Area "Spleen" applicable to this article?
Is the Subject Area "Microarrays" applicable to this article?
Is the Subject Area "Gene expression" applicable to this article?
Is the Subject Area "Immune response" applicable to this article?
Is the Subject Area "Amphibians" applicable to this article?
Animal models of infection
Is the Subject Area "Animal models of infection" applicable to this article?
Transcriptome analysis
Is the Subject Area "Transcriptome analysis" applicable to this article?


text:
Lotus Magellan war ein unter MS-DOS laufendes Desktop-Suchprogramm, das von Bill Gross entwickelt worden war und von der Lotus Development Corporation in den 1980er Jahren veröffentlicht wurde. Trotz seiner gerühmten Effektivität konnte sich Magellan auf dem Markt nicht behaupten, was möglicherweise auch an den zu dieser Zeit noch sehr kleinen Festplatten lag.

Magellan durchsuchte alle Dateien und Verzeichnisse eines Speichermediums (Festplattenlaufwerk oder Diskette) und erzeugte eine zentrale Indexstruktur, die es ermöglichte, die Dateien anzusehen ohne die einschlägigen Anwendungen zu starten, weil es die meisten zeitgenössischen Dateiformate lesen und interpretieren konnte.

Magellan war eine von mehreren maßgeblichen Programmen von Lotus Software (zum Beispiel Lotus 1-2-3, Lotus Notes und Bürosoftware für den Apple Macintosh), die es trotz ihrer anerkannten Nützlichkeit und beträchtlichen Marktanteile nicht verhindern konnten, dass die Firma im Jahre 1995 von IBM übernommen wurde. Die alte DOS-basierte Lotus-Software Magellan, Lotus Agenda, HAL (eine Erweiterung für Lotus 1-2-3) sowie Lotus Manuscript wurden mittlerweile als Freeware veröffentlicht.

Weblinks
 Lotus Magellan

Dateimanager
Magellan
DOS-Software
Ferdinand Magellan als Namensgeber

redpajama_set_name: RedPajamaWikipedia


Simply the best! A combination of vanilla, almond and butter with a citrus twist.
Our keylime cake is a spin on American's favorite pie. Filled with cream cheese icing and bits of graham cracker crust.
Homemade chocolate cake infused with coffee and filed with milk chocolate frosting. Our chocolate frosting pairs perfectly with our wedding vanilla cake.
Our classic sour cream almond cake with almond buttercream frosting.
Old fashioned banana cake made with ripe bananas and filled with caramel buttercream or cream cheese frosting.
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White chocolate cake filled with a light layer of buttercream and filled with a raspberry compote.
Most lemon cake paired with lemon buttercream or lemon cream cheese icing.
Homemade vanilla cake made with brown sugar and filled caramel buttercream frosting.


You can tidily store wood, garden furniture, recycling bins, garden equipment, lawnmowers, toys and more in the wood shed.
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How do we sort a string array in ascending or Descending Order in SAP Design Studio.
Jul 24, 2017 at 09:19 AM | 121 Views | Last edit Aug 02, 2017 at 05:05 PM 3 rev.
Just need help if anyone has faced this kind of scenario before. I want to sort Array of string elements in Ascending or descending order in Design Studio, considering Array has too many elements, so I cant specify order I want.
Considering example : var array_members=["xyz","mno","abc","def"]; I am trying to do it with loop statement. But I don't find any function to compare two string elements.
So can somebody help on this?


text:
Tag: Howard University
Howard Men's BBall cancels remainder of 2020-21 season
February 10, 2021 February 10, 2021 Brandon King
Courtesy of Howard University Athletic Media Relations WASHINGTON, D.C., Feb. 9, 2021 — The Howard University Department of Athletics has made the difficult decision to cancel the remainder of its 2020-21 men's basketball season out of an abundance of caution due to the health and safety concerns related to the COVID-19 pandemic. "We had several team members test positive in January, and were forced to place the program on hold, cancel several games and finally cancel our season. It was and remains the right thing for us to do," head men's…
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On the last day of bad old last year, Lake Superior State University released its 43rd annual List of Words Banished from the Queen's English for Misuse, Overuse and General Uselessness.
"Fake news" has to go not because there is no such thing, but because (a) people have the habit of calling real but unwanted or undesirable news "fake," and (b) because defining it isn't so easy.
"Fake news" is thus like bad yet award-winning art. You know it when you see it, but good luck developing an unambiguous definition.
This doesn't mean the complete disappearance of "fake news" from Facebook. On stories meeting their selection criteria for fakiness, they will link counter stories called "related articles." Call this the he-said-she-said approach.
They will soon learn that this is no easy task either. Discovering the truth in news reports is not only not simple. In many cases, it's not even possible.
Not that people aren't trying.
Some college kids think they have developed a browser plug-in that can alert users to "fake news." And a group of folks at the Fake News Challenge believe they can harness "artificial intelligence" (statistical models that have undergone dull-knifed plastic surgeries and name changes) to identify made-up stories with "an intention to deceive." Which is to say, propaganda.
It is charming the simple faith many have in the ability of computers to mimic human thinking. Yet all these algorithms can do is to note patterns in data, which when fed a new observation classifies it into one of the patterns. That means somebody has to create a list of news stories which have been without error or controversy placed into "truth" and "propaganda" bins.
Place a reactionary and progressive in a room and see if they can without disagreement classify any list of prominent news stories from, say, CNN. Since that can't be done, bias will and must be introduced into the algorithms.
This bias is fine, if your goal is like Facebook's, to disentrench "deeply held beliefs." But even then, no algorithm will classify perfectly; no, not even a deep-learning artificial intelligence neural quantum computer.
For one, there are sins of omission, which no algorithm can detect. How do you know, in advance, that a news agency should be covering a revolution when instead they are showing shots of a white van? You can't classify a non-existent story as "fake news," or as any kind of news.
Most limiting is that the algorithms do not work the way human thinking does. People grasp stories. We make mistakes, but we comprehend the words and meaning behind the words in a context beyond the ken of any computer. Computers are only mindless adding machines. Novelty will thus always stump any fake-news algorithm.
In the United States' current polarized political environment, the constant publishing of articles with vehemently opposing arguments has made it almost impossible for Google to rank information properly.
Let's say that this group believes Fact A and this group believes Fact B and you passionately disagree with each other and you are all publishing and writing about it and so forth and so on. It is very difficult for us to understand truth.
It isn't so easy for us humans, either. Which is why there are still disputes in, say, philosophy, even after thousands of years of trying to sort them out. And which is why there will always be disagreements between what is "fake news" and what isn't.


devilhorns closed T6947: Can't run enlightenment with wayland as user, a subtask of T7272: Merge support for elogind, as Resolved.
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We'd like to welcome everyone who've reached the semifinals of the FanFilm Awards 2017! Below is the list chosen by the fans and the judges. We begin voting for finalists now. Vote below for the respective category you wish to place your favorite fanfilms to be nominated for – titles without links have already been nominated for Best Script.
This is great. Congrats on the semi-finalists. I look forward to catching up on all these films!


Justice - R.F. Nariman, J.
PER : R.F. Nariman, J.
2) The present appeals raise an interesting question as to the correct interpretation of Section 2(22)(e) of the Income Tax Act, 1961, as amended in 1988.
The Assessee is a partnership firm consisting of three partners, namely, Mr. Naresh Goyal, Mr. Surinder Goyal and M/s Jet Enterprises Private Limited having a profit sharing ratio of 35%, 15% and 50% respectively. The Assessee firm had taken a loan of Rs. 28,52,41,516/- from M/s Jetair Private Limited, New Delhi. In this Company, the Assessee subscribed to the equity capital of the aforesaid Company in the name of two of its partners, namely, Mr. Naresh Goyal and Mr. Surinder Goyal totaling 48.19 per cent of the total shareholding. Thus Mr. Naresh Goyal and Mr. Surinder Goyal are shareholders on the Company's register as members of the Company. They hold the aforesaid shares for and on behalf of the firm, which happens to be the beneficial shareholder.
4) The question that arises in these appeals is as to whether Section 2(22)(e) of the Act gets attracted inasmuch as a loan has been made to a shareholder, who after the amendment, is a person who is the beneficial owner of shares holding not less than 10% of the voting power in the Company, and whether the loan is made to any concern in which such shareholder is a partner and in which he has a substantial interest, which is defined as being an interest of 20% or more of the share of the profits of the firm.
"8. The only surviving question is whether a loan advanced by a company to a H.U.F., which is the real owner of the shares, can be considered as a loan advanced to its shareholder. It is well-settled that an H.U.F. cannot be a shareholder of a company. The shareholder of a company is the individual who is registered as a shareholder in the books of the company. The H.U.F., the assessee in this case, was not registered as a shareholder in books of the company nor could it have been so registered. Hence, there is no gain-saying the fact that the H.U.F. was not the shareholder of the company. Mr. Sen did not contend otherwise.
Last edit: 1 year 2 months ago by amit.


The first own room for a baby should be amazing and unique. Do you agree? We collected for you 10 ideas on how the ceiling in the nursery can transform an ordinary room into the most favorite game world for your child!
It's not fresh idea, you could argue. But white ceiling is the perfect background for a spectacular lamps ensemble, for example, in the form of a sailing ship, balloon or moon.
The second most popular approach among designers of children's room is the sky with clouds on the ceiling.
Because exactly this natural background reflects the best lamps in the form of airplanes and small suns. If there is a rainbow there - cheerful mood for each day is guaranteed for your child!
Travel to the stars in your dreams and in reality! Your child will surely accept such a gift, when his/ her own room has no boundaries and any planet in our galaxy can be seen directly in the room, and stars will twinkle and mesmerize in the night!
Suitable supplement for a children's room in space style can be reduced model of our solar universe mounted in the high and of course - a telescope!
Do not forget that such a room should look fantastic especially at night - so add LED backlight simulating stars on the ceiling.
If you have small Mowgli or young Amazon growing at home - you can create in your children's room not just fabulous jungle, but a whole entertainment and sports complex with ladders, ropes and swings.
In order to visually increase the height of the ceiling designers often propose an extension of the theme of the wall to the ceiling: a picture or decorative object is in smooth transition from the wall to the ceiling.
It should be noted that this effect is most easily achieved by means of decoration of the walls and the ceiling (or part thereof) with stretched canvas with photo printing.
Desire to inherit the world from your favorite fairy tale or movie into the room is now quite feasible childhood dream! In addition, you can decorate not only ceiling, but all space too!
You can install for the little princess her own castle, for the pirate – his treasure's chest, for fans of science fiction and intergalactic travel - their own Pandora's box!
If your child is credited Jules Verne or admire the videos about marine life of Jacques-Yves Cousteau - make in his room something interesting in a nautical theme. This can be a ship cabin or a submarine.
And do not forget about your favorite marine adventure heroes of cartoons. You can try to recreate the world of the movie about Ariel Room for your girl.
Cats, dogs, lion, ferret, guinea pig, goldfish ... and even Tyrannosaurus. Which animal your kid likes?
A good idea to explore the animal kingdom with your child, and to transform his room into a natural habitat of his pet: funny farm, savannah, tropical island or marine depth.
What guy does not like sports cars? Regardless of age, a race shot or photo of red Ferrari will likely cause to your boy an overwhelming desire to grow quickly, to save up for his own car and get behind the wheel.
On the other hand, any other favorite vehicles can also become a focus of the interior design!
And if you want to create a night version of your babe's room - use LEDs for ceiling's decoration and phosphorescent paints on walls, which are visible only in the twilight and the night.


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San Angelo Chamber names Lynn Shipley 2020 Citizen of the Year
Lynn Shipley was named as the San Angelo Chamber of Commerce Citizen of the Year.
The announcement was made Thursday, Jan. 23, 2020 during the Chamber's annual banquet at the McNease Convention Center.
Shipley said he was proud of the work accomplished by the employees of the two San Angelo H-E-B stores and the people of San Angelo for helping keep the community a vibrant and prosperous place in West Texas.
"This is an incredible honor," Shipley said. "You always strive to do the very best that you can in all that you do. You never know how it's going to work out. All you can do is hope you made a positive impact on who you're working with and the folks you want to help. I've been given that honor in working with H-E-B. I can't stand up here this evening without complimenting both teams at both H-E-B stores. The folks of San Angelo are incredible. It's been an honor to work with you and serve you."
Shipley, the unit director of the original H-E-B, is a well-known figure around San Angelo, not only does he find ways to help those in need, he's often found working alongside volunteers, such as during the storm cleanup after the tornadoes that struck San Angelo and the area in spring 2019.
2019:Ronnie and Maria Hawkins named Citizens of Year by San Angelo Chamber of Commerce
2018:San Angelo Chamber of Commerce names Citizen of the Year
If you appreciate locally driven journalism, consider a digital subscription to GoSanAngelo.com. Follow us on Facebook, Twitter and Instagram for news updates. Submit news tips to News@GoSanAngelo.com.


research shows that early learning and care is essential to brain development in our young children, so we are guided along these lines to provide the best opportunities for your children in a nurturing environment. We view children as learners and they learn best through play which is a vital way for children to explore different ways of doing things and learn important lessons from their own hands on experience. With the guidance of our trained teachers the experiences provided promote and extend on your child's knowledge and skills so that they can always achieve!
children to grow and get a better understanding of the importance of culture and community.
We aim for children to become confident as independent learners giving them the opportunities to challenge themselves, problem solve, explore using their own natural curiosity, develop self help skills and risk taking in a caring, nurturing environment.
Our environments are rich in natural resources with space for children to run. They can interact with the environment and often are able to explore our community through walks and participation in community projects. We have great resources to extend the children's thinking with endless opportunities for learning.
Come and join our Kindergarten family where your child will develop skills and opportunities to embark on their life journey where learning will happen naturally as they enjoy what they are doing.


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Notebook: Youth Won't Change Game Plan
Head Coach Mike McCarthy is no stranger to starting rookies on offense.
Last year as the offensive coordinator of the San Francisco 49ers, McCarthy saw as many as seven rookies on his unit.
So it doesn't necessarily bother him that the Packers appear to be going into training camp with two rookies, second-round draft pick Daryn Colledge and third-rounder Jason Spitz, starting at guard on the offensive line. He doesn't expect the youth on the interior of the line to alter any game plans.
"If they're in a starting position I'm comfortable with it because they've earned that opportunity," McCarthy said. "If you have so many (rookies), it's not very comfortable. You have to call a different game and your approach is different.
"But I think regardless of who our starting guards are, we're going to be young but I don't think it's going to change the way we're trying to play. As long as we're not limited because we are so young, I think we'll be fine."
McCarthy has been particularly impressed with Spitz, who he says shows tremendous discipline with his hands for a young player. Spitz, from Louisville, also has been asked to play multiple positions on the line during practice and acquitted himself well.
But the starting positions are by no means settled. Second-year pro Junius Coston, a fifth-round pick in 2005, is also among those in the mix at guard.
"I think we have very healthy competition there with all those young guys, and Junius is definitely a big part of that," McCarthy said. "The good news is they all have a tremendous upside, so we have to get to that as quick as we can."
Depth improving
Asked if anything has been a pleasant surprise to him during the organized team activities (OTAs), McCarthy pointed to the depth of various position groups.
"I think we've got a lot more depth than I realized before we hit the field, particularly in the secondary and defensive line," he said. "I think our linebacker group is really coming together, ... and the tight end group is the most athletic group I've been around since I've been in the league."
That's saying something for the tight ends, a group that includes veterans Bubba Franks, David Martin and Donald Lee. Back in 2003 with McCarthy coordinating the Saints' offense, tight ends Boo Williams and Ernie Conwell combined for 74 catches, 726 yards and seven TDs.
Favre to stay in shape
Quarterback Brett Favre did not participate in the final OTA, but McCarthy indicated he'll get a personal visit from strength and conditioning coach Rock Gullickson in Mississippi in the next month to work on any conditioning that's needed.
As far as learning a new version of the West Coast offense, McCarthy said Favre has made the transition smoothly and would expect him to hit the ground running with the offense when training camp begins.
"He has excellent recall. I don't anticipate any problems there," McCarthy said. "I just think it's an old dog with new tricks. He's done it so long one way, and we've jolted him a little bit with some new learning, but frankly I told him I think it's healthy for him."
McCarthy also felt Favre was at his best thus far in the offseason right before he left on Tuesday afternoon.
"As you watched him go through OTAs, to me you saw his arm coming around more and more," he said. The last couple practices it was at the point where he's almost showing off, sticking it in there when he probably shouldn't be. But he can.
"You definitely saw the ball starting to spin off his hand with a lot of velocity this past week."
In addition to the players, the coaching staff will be taking time off as well prior to the start of training camp.
McCarthy's assistant coaches are expected to be off on Friday, returning the week before camp begins. McCarthy plans to leave Green Bay as well but confessed there's plenty of work for him to stay on top of between now and July 28.
"I'll be getting away, but I don't know if I'll be off," he said with a smile.
"I'll be spending time with my daughter. This is time of year we spend our quality time together, and that won't change."


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Stuck Disneyland Ride Leads to $8,000 in Damages
Jul 22, 2013 by Donald Scarinci	in Law
Some of the cases attorneys take never cease to amaze me. The fact that they win some of the more outlandish ones amazes me even more.
Anyone who has taken the "Small World" ride at Disneyland knows that it sometimes takes days to get the tune of the song, "It's a Small World," out of your head. It is little wonder that someone would want to sue for pain and suffering as a result of having to listen to that song for a long time.
Recently, a disabled man trapped on the Disneyland ride did just that. He sued for pain and suffering caused by the constant repetition of the "Small World" tune. He won a judgment of $8,000 in damages.
According to the lawsuit against Disney, Jose Martinez visited the amusement park in 2009 with his wife. While they were on the ride "It's a Small World," it began to suffer mechanical problems. While other riders were quickly evacuated, Martinez was forced to wait for over 30 minutes while the ride's music continued to play.
Because Martinez is paralyzed and uses a wheelchair, Disney staff told him that they could not move him. Instead, they waited for the rise to be fixed. Martinez required medical attention once he finally exited the ride. According to the lawsuit, he suffered from dysreflexia, "a condition experienced by those with spinal cord injuries caused by stress and overstimulation."
Martinez ultimately filed suit. Among other claims, he alleged that Disney should have contacted the fire department to evacuate him as soon as the ride broke down. He also maintained that Disney failed to warn disabled passengers prior to boarding that they could become trapped if the ride malfunctioned. Meanwhile, Disneyland maintained that it provided "all appropriate assistance."
Ultimately, the court agreed with the plaintiff on the theory of premises liability. Disney "should have known that there was a likelihood of harm to a disabled person, as reflected in its own standards for that ride," the judge ruled. Martinez was awarded $4,000 for pain and suffering and $4,000 for other disability access violations.
For more interesting Law cases, check out my Law Category in my favorites section. I particularly like "Who wants to be a millionaire." If you are interested in speak with an attorney, please visit my law firm www.scarincihollenbeck.com.
Tagged with: "Small World", damages amusement park, disability lawsuit, disneyland lawsuit
About the author Donald Scarinci
Donald Scarinci is the Founding Partner of Scarinci Hollenbeck. He writes and lectures extensively about Constitutional Law and edits the award winning, The Constitutional Law Reporter. His practice focuses on representing public institutions and businesses that interact with government. You can read his full bio at ScarinciHollenbeck.com
Connect with Donald Scarinci
Columns by Donald Scarinci
Insider NJ
Politicker NJ
The Frelinghuysen Family is NJ History
16 March 2017 - Donald Scarinci
The Frelinghuysen's are one of New Jersey's oldest and most important families. Over the course of more than 200 years, the family has produced four United States senators and two members of the House of Representatives. Rep. Rodney Frelinghuysen (R-NJ) has served 11 terms in the House. He serves 28 Morris County municipalities and also represents parts[...] The post The Frelinghuysen Family is NJ History appeared first on Insider NJ. [...]
Brian Stack Protects Sanctuary Cities from Federal Funding Cuts
8 March 2017 - Donald Scarinci
State Senator and Union City Mayor Brian Stack is taking the lead with other NJ State Legislators on State legislation that would lessen the blow of President Trump's executive order on immigration. Senate Bill Number 3007 would establish a program to provide funding to municipalities that have lost federal grant funding because they are a[...] The post Brian Stack Protects Sanctuary Cities from Federal Funding Cuts appeared first on Insider NJ. [...]
Proposed Bill Adds New OPRA Exception
27 February 2017 - Donald Scarinci
New Jersey lawmakers are currently considering legislation that would add a new exception to the state's Open Public Records Act (OPRA). Unlike broad attempts to overhaul the state's public record law, Assembly Bill Number 4532 focuses on a very narrow issue — preventing businesses from using OPRA requests for marketing purposes. Spirit of OPRA New[...] The post Proposed Bill Adds New OPRA Exception appeared first on Insider NJ. [...]
Trump's Business Ties and the Constitution's Emoluments Clause
The legality of President Donald Trump's vast business ties is governed by a clause of the U.S. Constitution that most Americans have never heard of and few legal scholars agree on. The clause prohibits government officers from accepting emoluments, which is a fancy way of referring to money or something else of value, from foreign[...] The post Trump's Business Ties and the Constitution's Emoluments Clause appeared first on Insider NJ. [...]
Bill Gates Was Wrong About the Pandemic in 2020—Here's What He Thinks Now
For the pandemic to really end, we will need long-lasting vaccines that can prevent re-infection, Gates said. [...]
'The Book of Boba Fett' Chapter 3 Recap: This Crime Lord Is a Little Too . . . Nice?
Even Wookie assassin Black Krrsantan can't keep things from getting more than a little dull as Boba Fett models a kinder, gentler kingpin [...]
World's First Commuter Electric Plane Is Preparing for Maiden Flight
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Alex Rodriguez Is Heading Back to Central Park West
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Volkswagen vows to double electric vehicle sales in China in 2022. [...]
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How Luxury Brands Are Celebrating Lunar New Year 2019, The Year Of The Pig
Barry Samaha Contributor
Gucci advertisement of a dedicated collection that celebrates Chinese New Year 2019, the Year of the Pig.
Gucci; The Three Little Pigs © Disney
Luxury brands are all on board for Lunar New Year. Though not as widespread and celebrated in Western cultures, it does rank high in China—a market where consumers still pay full price for expensive wares. According to a report by Bain & Company, China represents "32% of the global luxury consumption." Indeed, the commercial viability of this region, one that is still growing year over year, is not to be taken lightly. This is why major companies are consistently offering capsule collections that feature characters found in the Chinese zodiac.
In 2018, the year of the dog, French, American and Italian labels cast the canine on a variety of bags, jewelry and sweaters. The year before that, the rooster was the star attraction. These animals are attractive; they're beloved. They can easily be plopped on accessories or apparel and enhance the overall look. The 2019 symbol, however, is a not-so-easy sell. It is the year of the pig, and though some have affinity for the barnyard mammal, it doesn't have the same universal draw as its predecessors—read: there's a reason why calling someone a pig has negative connotations.
That said, some of the biggest brands in the luxury business are undeterred by the pig's less-than-stellar reputation. They have either enhanced its adorable qualities or found cherished characters from the past and reinterpreted them for today.
Check out five luxury labels that have put their spin on the year of the pig.
For the Lunar New Year, Gucci looked to The Walt Disney Company for inspiration. The title characters in Three Little Pigs, the animated short film released in 1933, are cast on number wallets, bags and sweaters. Indeed, cutesy prints fall right in line with Gucci's new direction under its designer Alessandro Michele, who transformed the once-ultra-sexy company into one that glamorizes peculiarities—like Disney cartoons on a six-figure pullover with racing stripes.
Gucci Sweatshirt with Three Little Pigs, $1,100, Gucci.com.
Moschino also looked to an animated character for Chinese New Year. Warner Brothers's Porky Pig, the beloved stuttering swine, and Petunia Pig, his girlfriend, pose on a collection of streetwear staples: backpacks, hoodies and denim jackets. The brand's designer Jeremy Scott is a master at kitsch who walks the fine line between tony and tawdry—and this capsule line certainly fits into this credo.
Moschino Chinese New Year Clutch and Backpack, $875, Moschino.com.
On the opposite side of the kitsch spectrum is Vacheron Constantin. The Switzerland-based watch company is celebrated for classic styles, fine finishes and technical mastery—attributes that describe its offering for the Lunar New Year. For its Year of the Pig watch, which is part of the Métiers d'Art The Legend of the Chinese Zodiac collection, the brand uses enameling, gems-setting and other artistic aspects of watchmaking. The rendering of the piglet on the dial is a detailed relief etching done on either pink gold or platinum. To whit: it's definitely a prize pig and not a porker.
Vacheron Constantin Métiers d'Art The Legend of the Chinese Zodiac – Year of the Pig Watch in 18K Pink Gold, $102,000, Vacheron-constantin.com.
From lions and elephants to pandas and porcupines, Roberto Coin's Animalier collection is a comprehensive menagerie. And the most adorable one in the jeweler's stable is the Piglet ring. Set in 18K gold with bold pink sapphires, the piece will surely have pig fans squealing with delight.
Roberto Coin Animalier Piglet Ring in 18K White Gold with Pink and Blue Sapphires, $18,500. Robertocoin.com.
Playing up the adorable qualities of baby pigs seems to be the modus operandi for brands offering items this Lunar New Year—and Louis Vuitton is no different. The French luxury company has a collection of accessories, including a pink scarf with adorable graphics of abstract piglets.
Louis Vuitton Superstition Square Scarf, $370, Louisvuitton.com.
For More, See:
Valentine's Day Gift Guide 2019: The Best Luxury Earrings Of The Season
Valentine's Day Gift Guide 2019: The Best Luxury Bracelets Of The Season
Commit to Your 2019 New Year's Resolutions With These Chic Shopping Finds
Follow Barry Samaha on Twitter (@barry_samaha) and Instagram (@barrysamaha).
Barry Samaha
As a lifestyle writer and market editor, I like to keep abreast of the newest and greatest in the worlds of fashion, beauty and home décor. I live in New York City and c...