Abstract
The global amphibian biodiversity crisis is driven by disease, habitat destruction and drastically altered ecosystems. It has given rise to an unprecedented need to understand the link between rapidly changing environments, immunocompetence and wildlife health (the nascent field of ecoimmunology). Increasing our knowledge of the ecoimmunology of amphibians necessitates the development of reliable, field-applicable methods of assessing immunocompetence in non-model species. The phytohaemagglutinin (PHA) inflammation assay uses a lectin to elicit localized inflammation that reflects an organism's capacity to mount an immune response. Although extensively used in birds to assess responses to environmental change, stress and disease, its application in amphibians has been extremely limited. We developed, validated and optimized a practical and effective phytohaemagglutinin inflammation assay in phylogenetically distant amphibians and demonstrated its suitability for use in a wide range of ecoimmunological studies. The protocol was effective for all species tested and worked equally well for both sexes and for adult and sub-adult animals. We determined that using set-force-measuring instruments resulted in a 'compression effect' that countered the inflammatory response, reinforcing the need for internal controls. We developed a novel method to determine peak response times more accurately and thereby improve assay sensitivity. Histological validation demonstrated considerable interspecies variation in the robustness of amphibian immune defences. Importantly, we applied the assay to a real-world scenario of varying environmental conditions and proved that the assay effectively detected differences in immune fitness between groups of animals exposed to ecologically meaningful levels of density stress. This provided strong evidence that one cost of metamorphic plasticity responses by tadpoles to increasing density is a reduction in post-metamorphic immune fitness and that metamorphosis does not prevent phenotypic carry-over of larval stress to the adult phenotype. This assay provides an effective tool for understanding the role of global environmental change in the amphibian extinction crisis.
Highlights
Recent, resurgent interest in the effects of environmental stress on organisms has been precipitated by the broader biodiversity crisis, which has its roots in anthropogenically driven global environmental change
There was a further increase in leg thickness between 12 and 24 h postinjection in L. aurea (t1,82.6 = 4.52, P < 0.001; Fig. 4E). Both tree frogs reached peak response by 24 h post-injection, which was a maximum of 7.64% increase in leg thickness for L. aurea and 4.41% for L. revelata
Our study demonstrated that the PHA assay is effective across a range of phylogenetically distant anuran amphibian taxa and, importantly, that it is capable of detecting differences in immunocompetence between groups of animals that have been stressed in changing environmental conditions that are ecologically meaningful
Summary
Resurgent interest in the effects of environmental stress on organisms has been precipitated by the broader biodiversity crisis, which has its roots in anthropogenically driven global environmental change. Global environmental change has been associated with rapid adjustments to environments through threatening processes, such as habitat destruction, invasive species, emerging infectious diseases and climate change (Reich et al, 2012; Pauls et al, 2013; Sutherland et al, 2013). The link between environment, immunocompetence and organismal health has received increasing attention in the fields of ecology and conservation, sparking rapid growth in the newly emerged field of ecoimmunology (Ros et al, 1997; Saino et al, 1997; Zuk and Johnsen, 1998; Acevedo-Whitehouse and Duffus, 2009; Rollins-Smith and Woodhams, 2012; Pigeon et al, 2013)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
