Recently, Drs. Ellison, Zamudio, Lips, and Muletz-Wolz published their work focused on some of the ways amphibians respond to an infection by Batrachochytrium dendrobatidis (Bd). Bd is a fungus that is causing devastating worldwide decline of amphibians, meaning that understanding how some species manage the infection is important for conservation of myriad species. Using an elegant experimental set up and subsequent RNA sequencing data, Dr. Ellison and co-authors suggests that the variation in amphibian susceptibility to the fungus, which is related to temperature, occurs due concurrent temperature-dependent shifts in immune system function; lower temperatures were associated with an inflammatory response while higher temperatures with an adaptive immune response. Understanding exactly how and when this fungus alters wild amphibian populations is important for conservation of these often imperiled species. For more information, please see the full article and the interview with Dr. Ellison below. 

What led to your interest in this topic / what was the motivation for this study? I have always been fascinated with how parasites and pathogens influence fitness and shape host populations, particularly generalists infecting a wide range of host species. The pathogenic chytrid, Batrachochytrium dendrobatidis (Bd), is arguably one of the most generalist pathogens known to science, capable of infecting hundreds of amphibian species globally. However, even within a single host species, disease outcome (e.g. succumbing to or clearing infection) is highly variable and is often temperature-dependent. Given the devastating impacts Bd has already had on amphibian populations, the recent discovery of another amphibian-killing chytrid (B. salamandivorans), and the ever-pressing threat of climate change, we were driven to uncover how amphibian gene expression responses to chytrid infections vary under different temperatures.

What difficulties did you run into along the way? For me, it was the sheer scale of the sequencing dataset. Plethodon salamanders, notorious for their large genome sizes, had yet to have a published genome or transcriptome to use as a reference for RNAseq studies such as ours. Therefore, we had to ensure sufficient sequencing to de novo assemble the transcriptome, and enough per-sample depth to capture potentially subtle but important changes in gene expression due to temperature and infection. With multiple temperature treatments and multiple disease outcomes at each temperature, this resulted in relatively large RNAseq dataset of over 2 billion reads. Thankfully, having returned to Wales from the US by the time we received our sequence data, I had access to Supercomputing Wales, a nationwide high-powered computing initiative that allowed me to handle the computationally intensive analyses. More importantly, without the hard work of the other authors to carefully design and execute the highly-controlled animal experiments to generate the tissue samples, this study would simply not be possible.

What is the biggest or most surprising innovation highlighted in this study? I think that, within a relatively narrow thermal range, the substantial shifts in the types of immune genes being expressed in response to infection is really important to our understanding chytrid infection dynamics. The finding that adaptive immune transcripts (particularly those involved in MHC pathways) are more highly expressed at warmer temperatures – where amphibians tend to survive infection better – is most exciting. Given the growing evidence for the importance of certain MHC allele variants in Bd resistance, our results suggest it is not only be what MHC genotype amphibians possess, but how they express them during infection that dictates survival.

Moving forward, what are the next steps in this area of research? This study, while providing new insights into how temperature influences Bd-amphibian interactions, has generated many further questions. Some of the authors on this study have recently shown both temperature and Bd has a significant impact amphibian skin microbiome communities, a potentially critical line of defense against infections. It is currently unknown whether temperature-dependent host immune expression responses to Bd shapes skin microbiomes during infection or if skin bacteria are influencing host responses (or a combination of both). Work to directly assess host gene expression under different microbial community compositions would be an exciting future avenue of research. In addition, further investigation of both MHC genotype and expression phenotype simultaneously could be highly relevant to understanding intraspecific variation in chytrid resistance. Finally, we have previously developed methods to quantify Bd gene expression in vivo; it would be fascinating to couple our current findings with how Bd genes are expressed in-host under different temperatures.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? Many others on this blog have already highlighted the importance of well thought out experimental designs, and the need to grips with the theory before embarking on a project, that I can only echo. Although having now worked on many transcriptomic datasets in non-model organisms, I still sometimes get overwhelmed with the amount of information that could be potentially conveyed in a manuscript, particularly with more complex experimental designs such as this study. I recommend periodically taking a step back from your analyses, share it with colleagues to gauge the most important “headline” results, and finally, don’t worry that some things have to go as supplemental material; they can still be gems of information that kick-off an exciting new line of inquiry for someone!

What have you learned about methods and resources development over the course of this project? With high-throughput sequencing methods becoming ever more accessible and the explosion of innovative ways to analyse and present NGS data, it is all too easy to feel your project is not “cutting-edge” enough. It’s all very well having billions of sequences and a slick set of figures, but a research team most importantly needs to be able to provide meaningful biological/ecological interpretation. That’s why it has been great to be part of a collaborative team of amphibian ecologists and geneticists, which was critical to the development of this new resource of information on salamander transcriptomic responses to temperature and infection.

Describe the significance of this research for the general scientific community in one sentence. The thermally-altered transcriptional responses of salamanders to fungal pathogen infection is an important component to understanding observed seasonal and climatic patterns of chytrid disease outbreaks. 

Describe the significance of this research for your scientific community in one sentence. Our results suggest shifts from inflammatory to adaptive immune gene expression responses to Bd infection at warmer temperatures are a key component to thermal and/or seasonal patterns of amphibian chytridiomycosis.

Ellison A, Zamudio K, Lips K. Muletz-Wolz C. 2019. Temperature-mediated shifts in salamander transcriptomic responses to the amphibian-killing fungus. Molecular Ecology 28:50586-5102.

The color variation that exists among individuals has lent itself to the study of selection since Darwin. Recently, Zaman, Hubert, and Schoville (2019) investigated the effects of selection on the diversity of the wing color pattern in the butterfly Parnassius clodius across a large portion of its range. These researchers found evidence supporting the idea that coloration may serve as a warning signal to predators, providing some predator avoidance benefits to individuals. In addition, the variation of solar radiation and precipitation observed geographically across sites was negatively correlated with the amount of melanin observed at each site. This suggests that the occurrence of melanin may provide a selective advantage in the form of thermoregulatory function. For more information on how selection influences butterfly wing coloration, please see the full article and the interview with Dr. Schoville below. 

What led to your interest in this topic / what was the motivation for this study? Khuram and I were both interested in butterfly color pattern variation, and in particular, cases where there might be competing selective pressures acting on wing pattern phenotypes. Most work on butterfly wing patterns focuses on predator-prey interactions and aposematic colors, but butterfly wings are essential to flight performance and important in thermal regulation. A number of recent papers have shown that butterfly color pattern appears to be responding to climate warming, and then there are well-known cases (such as alpine Colias, thanks to Ward Watt) where thermoregulation has been linked to basking behavior and the pigment on wings. Thus, in examining variation in Parnassius clodius (which occurs over a broad elevation and latitudinal range), we hoped that we could decouple environmental signals that might act on different wing color elements. 

What difficulties did you run into along the way? Sampling our butterflies across this large region was a major challenge, particularly as adult flight times are rather short (two to three weeks). And then we were surprised by the strong difference in adult phenology across sites (some adults are active in May, others in late July). This is evident regionally (Utah versus Washington), and across elevation within a region. In the end, it required three years of effort, with some very long road trips from Wisconsin.

What is the biggest or most surprising innovation highlighted in this study? After some initial efforts to link aposematic variation (red eyespots) indirectly to predator communities (through climate variables that might covary with predator abundance), we realized this was too tenuous. So, we were delighted to discover publicly available data on bird abundance. While this did not solve the problem (perhaps due to lack of spatial resolution in the bird data), I think using this data to analyze butterfly wing patterns was one of the more innovative aspects of our paper. We had a much easier time linking spatial climate data to melanization (dark pigmentation). As an aside, this raises the important point that some data, i.e. abiotic environmental data, is much easier to come by than biotic data. This is unfortunate, as we expect biotic selective forces to be equally or more important drivers of microevolution in some cases.

Moving forward, what are the next steps in this area of research? We’d like to extend this work in two directions. First, we’d like to connect color pattern traits to underlying genes and measures of heritability. Although the genes controlling butterfly color pattern are well studied, to date no representative of the snow Apollo subfamily Parnassinae have been included in these efforts. Members of the family are tremendously variable and quite stunning in their dramatic contrasts of color. Second, experiments are needed to link our inferences of ecological selection to fitness differences, as well as performance in the field. Physiological assays of melanic variants, coupled with mechanistic thermodynamic models, have been developed for Colias butterflies (see Joel Kingsolver and Lauren Buckley’s work). This type of modeling could provide important connections to conservation of Parnassius clodius populations under changing climates, and might perhaps extend to conservation work on other highly threatened Parnassius species.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? The development of novel approaches is a key part of advancing biological knowledge, but it can be a daunting endeavor given the breadth and scope of the scientific literature nowadays. Integrating multiple approaches, on the other hand, can equally help to advance our knowledge and provide opportunities to address long-standing questions. This is the direction we took in this paper. My personal view is that students should to try to master multiple techniques (assemble a toolkit, so to speak) and apply those techniques to fundamental problems. Hopefully, it’s a lot of fun in the process and leads to interesting collaborations!

What have you learned about methods and resources development over the course of this project? We are entering a golden age of data-rich resources, in terms of spatial environmental data and genomics data. These increasingly provide the power to test refined hypotheses about evolutionary and ecological processes, and are becoming more accessible to all researchers. One of my favorite accomplishments in the paper is using genetic covariance data among populations (relatedness data) as a covariate in fitting morphology ~ environment models. The use of such population contrasts is important in controlling for non-independence in the data due to ancestry. While we have known about the importance of genetic covariance in hypothesis testing for some time (thanks to Joseph Felsentstein’s work), it is only recently possible to use genome-wide data. This provides very precise measures that are highly informative, and enabled us to rule out the role of genetic drift as a driver of wing pattern variation. 

Describe the significance of this research for the general scientific community in one sentence. Our research demonstrates that butterfly wing color patterns evolve in response local climate conditions, as a way to regulate body temperature.

Describe the significance of this research for your scientific community in one sentence. Our work demonstrates that elements of butterfly wing pattern phenotypes respond independently to different sources of selection, with climate variation acting on thermoregulatory ability as an important driver of butterfly color pattern.

Zaman K, Hubert MK, Schoville SD. 2019. Testing the role of ecological selection on color pattern variation in the butterfly Parnassius clodius. Molecular Ecology 28:50586-5102.