Analysis of the transcriptome of the needles and bark of Pinus radiata induced by bark stripping and methyl jasmonate

Background Plants are attacked by diverse insect and mammalian herbivores and respond with different physical and chemical defences. Transcriptional changes underlie these phenotypic changes. Simulated herbivory has been used to study the transcriptional and other early regulation events of these plant responses. In this study, constitutive and induced transcriptional responses to artificial bark stripping are compared in the needles and the bark of Pinus radiata to the responses from application of the plant stressor, methyl jasmonate. The time progression of the responses was assessed over a 4-week period. Results Of the 6312 unique transcripts studied, 86.6% were differentially expressed between the needles and the bark prior to treatment. The most abundant constitutive transcripts were related to defence and photosynthesis and their expression did not differ between the needles and the bark. While no differential expression of transcripts were detected in the needles following bark stripping, in the bark this treatment caused an up-regulation and down-regulation of genes associated with primary and secondary metabolism. Methyl jasmonate treatment caused differential expression of transcripts in both the bark and the needles, with individual genes related to primary metabolism more responsive than those associated with secondary metabolism. The up-regulation of genes related to sugar break-down and the repression of genes related with photosynthesis, following both treatments was consistent with the strong down-regulation of sugars that has been observed in the same population. Relative to the control, the treatments caused a differential expression of genes involved in signalling, photosynthesis, carbohydrate and lipid metabolism as well as defence and water stress. However, non-overlapping transcripts were detected between the needles and the bark, between treatments and at different times of assessment. Methyl jasmonate induced more transcriptional responses in the bark than bark stripping, although the peak of expression following both treatments was detected 7 days post treatment application. The effects of bark stripping were localised, and no systemic changes were detected in the needles. Conclusion There are constitutive and induced differences in the needle and bark transcriptome of Pinus radiata. Some expression responses to bark stripping may differ from other biotic and abiotic stresses, which contributes to the understanding of plant molecular responses to diverse stresses. Whether the gene expression changes are heritable and how they differ between resistant and susceptible families identified in earlier studies needs further investigation.

Interactive effects of biotic stressors and provenance on chemical defence induction by holm oak (Quercus ilex)

Key Message The patterns of induced chemical defences in Quercus ilex leaves are specific to the biotic stress factor that causes them. Interactive effects between stressors depend on provenance. Abstract Quercus forests are suffering serious decline worldwide, closely linked to the consequences of climate change. The increase of biotic stressors threatens the survival of the holm oak (Quercus ilex), a dominant tree species in the Mediterranean Basin. A better understanding of its resistance mechanisms is urgently required to enable a better control of its decline. In this work, the ability of holm oaks from six Iberian provenances to respond to multiple biotic damage is studied through an analysis of their induced chemical defence patterns. Using 2016 seedlings established in a common garden trial (6 regions × 12 families/region × 7 seedlings/family × 4 treatments), biotic damage was induced at the root level (by infection with the widespread pathogen Phytophthora cinnamomi) and at the above-ground level (by mechanical defoliation). The levels of constitutive and induced total phenols, total tannins and condensed tannins were measured. Results showed that (1) the defensive chemical patterns present significant local and geographical variation, (2) survival to stress is more related to constitutive defences than induced ones, (3) the induced response is stressor-specific, and (4) there is an interactive effect amongst stressors whose sign (induction/inhibition) depends on the provenance. These findings on biotic stressor effects on the chemical defences and survival of holm oak can contribute to the development of genetic material selection programs in the integrated control of the widespread decline of Quercus.

Do Active and Passive Antipredator Defences in the Toad Epidalea calamita Differ between Males and Females from Natural Habitats and Agrosystems?

Due to their assumed costs, simultaneous antipredator strategies are expected to face trade-offs, which, however, could be milder in individuals subjected to a more intense predator pressure. In this work, I studied the relationship between locomotion and parotoid glands in the natterjack toad, Epidalea calamita. Specifically, I predicted that individuals with reduced sprint speed would rely more on their chemical defences, having larger and more aposematically coloured parotoid glands. In addition, I expected this trade-off to be more evident in females and toads from pine grove habitats, because, according to previous work, males and toads from agrosystems are under greater predator pressure. However, sprint speed showed no relationship with coloration, but toads with proportionally greater parotoid glands were also proportionally faster. Thus, the costs of these antipredator traits might not be high enough to make them interfere, or the benefits of simultaneous optimisation of sprint speed and parotoid gland size might outweigh the costs of it in some individuals. In any case, habitat and sex did not affect these relationships, so the trends detected are valid across sexes and the habitats studied.

DNA metabarcoding reveals host-specific communities of arthropods residing in fungal fruit bodies

Biological communities within living organisms are structured by their host's traits. How host traits affect biodiversity and community composition is poorly explored for some associations, such as arthropods within fungal fruit bodies. Using DNA metabarcoding, we revealed the arthropod communities in living fruit bodies of eleven wood-decay fungi from boreal forests and investigated how they were affected by different fungal traits. Arthropod diversity was higher in fruit bodies with a larger surface area-to-volume ratio, suggesting that colonisation is crucial to maintain arthropod populations. Diversity was not higher in long-lived fruit bodies, most likely because these fungi invest in physical or chemical defences against arthropods. Arthropod community composition was structured by all measured host traits, namely fruit body size, thickness, surface area, morphology and toughness. Notably, we identified a community gradient where soft and short-lived fruit bodies harboured more true flies, while tougher and long-lived fruit bodies had more oribatid mites and beetles, which might reflect different development times of the arthropods. Ultimately, close to 75% of the arthropods were specific to one or two fungal hosts. Besides revealing surprisingly diverse and host-specific arthropod communities within fungal fruit bodies, our study provided insight on how host traits structure communities.

Cascading effects of caffeine intake by primary consumers to the upper trophic level

Secondary metabolites are central to understanding the evolution of plant–animal interactions. Direct effects on phytophagous animals are well-known, but how secondary consumers adjust their behavioural and physiological responses to the herbivore's diet remains more scarcely explored for some metabolites. Caffeine is a neuroactive compound that affects both the behaviour and physiology of several animal species, from humans to insects. It is an alkaloid present in nectar, leaves and even sap of numerous species of plants where it plays a role in chemical defences against herbivores and pathogens. Caffeine effects have been overlooked in generalist herbivores that are not specialized in coffee or tea plants. Using a host–parasitoid system, we show that caffeine intake at a relatively low dose affects longevity and fecundity of the primary consumer, but also indirectly of the secondary one, suggesting that this alkaloid and/or its effects can be transmitted through trophic levels and persist in the food chain. Parasitism success was lowered by ≈16% on hosts fed with caffeine, and parasitoids of the next generation that have developed in hosts fed on caffeine showed a reduced longevity, but no differences in mass and size were found. This study helps at better understanding how plant secondary metabolites, such as caffeine involved in plant–animal interactions, could affect primary consumers, could have knock-on effects on upper trophic levels over generations, and could modify interspecific interactions in multitrophic systems.

First characterization of toxic alkaloids and volatile organic compounds (VOCs) in the cryptic dendrobatid Silverstoneia punctiventris

Background Poison frogs are known for the outstanding diversity of alkaloid-based chemical defences with promising therapeutic applications. However, current knowledge about chemical defences in Dendrobatoidea superfamily has two sources of bias. First, cryptic, brown-colored species have been neglected in comparison to those conspicuously colored, and second, there has been little interest in characterizing metabolites other than alkaloids mediating defensive functions. In an effort to contribute to fill the gap of knowledge about cryptic species and broadening the spectrum of compounds analyzed we have applied head-space solid phase microextraction coupled to gas chromatography and mass spectrometry (HS-SPME/GC-MS) for extracting amphibian alkaloids and volatile organic compounds (VOCs) from Silverstoneia punctiventris. Results Using the skin from 8 specimens in 4 biological replicates we have found 33 different compounds. Twenty of them were classified as VOCs into 15 chemical classes including alkanes, alcohols, carbonyl compounds, methylpyridines, benzothiazoles, N-alkylpyrrolidines, pyrazines, and sesquiterpenoids, some of which were previously reported as repellents, defence compounds or defence pheromones in other organisms, and as sex pheromones in a treefrog. Interestingly, six of the remaining compounds were identified as alkaloids previously reported in other toxic/unpalatable dendrobatid frogs. Conclusions This is the first report of alkaloids and VOCs found in the Silverstoneia genus, which has been assumed for decades as non-chemically defended. This study establishes HS-SPME/GC-MS as a new application for a simultaneous approach to amphibian alkaloids and VOCs in poison frogs while opens up new research questions to assess the co-occurrence of both type of compounds and to investigate the evolutionary significance of a defence gradient that includes olfactory avoidance, unpalatability, and toxicity in dendrobatids. In addition, our results show that amphibian alkaloids could have a dual function (olfactory at distance, taste by contact) never explored before neither in Silverstonaeia nor in any other dendrobatid species.

Shifting chemical defence or novel weapons? A review of defence traits in Agarophyton vermiculophyllum and other invasive seaweeds

Seaweed bioinvasions increasingly affect coastal environments around the world, which increases the need for predictive models and mitigation strategies. The biotic interactions between seaweed invaders and invaded communities are often considered a key determinant of invasion success and failure and we here revise the current evidence that the capacity of seaweed invaders to deter enemies in newly reached environments correlates with their invasion success. Particularly efficient chemical defences have been described for several of the more problematic seaweed invaders during the last decades. However, confirmed cases in which seaweed invaders confronted un-adapted enemies in newly gained environments with deterrents that were absent from these environments prior to the invasion (so-called “novel weapons”) are scarce, although an increasing number of invasive and non-invasive seaweeds are screened for defence compounds. More evidence exists that seaweeds may adapt defence intensities to changing pressure by biological enemies in newly invaded habitats. However, most of this evidence of shifting defence was gathered with only one particular model seaweed, the Asia-endemic red alga Agarophyton vermiculophyllum, which is particularly accessible for direct comparisons of native and non-native populations in common garden experiments. A. vermiculophyllum interacts with consumers, epibionts and bacterial pathogens and in most of these interactions, non-native populations have rather gained than lost defensive capacity relative to native conspecifics. The increases in the few examined cases were due to an increased production of broad-spectrum deterrents and the relative scarcity of specialized deterrents perhaps reflects the circumstance that seaweed consumers and epibionts are overwhelmingly generalists.

Exposure to Batrachochytrium dendrobatidis affects chemical defences in two anuran amphibians, Rana dalmatina and Bufo bufo

Background Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). Results Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. Conclusions The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events.