Internal state dynamics shape brainwide activity and foraging behaviour.

The brain has persistent internal states that can modulate every aspect of an animal's mental experience1-4. In complex tasks such as foraging, the internal state is dynamic5-8. Caenorhabditis elegans alternate between local search and global dispersal5. Rodents and primates exhibit trade-offs between exploitation and exploration6,7. However, fundamental questions remain about how persistent states are maintained in the brain, which upstream networks drive state transitions and how state-encoding neurons exert neuromodulatory effects on sensory perception and decision-making to govern appropriate behaviour. Here, using tracking microscopy to monitor whole-brain neuronal activity at cellular resolution in freely moving zebrafish larvae9, we show that zebrafish spontaneously alternate between two persistent internal states during foraging for live prey (Paramecia). In the exploitation state, the animal inhibits locomotion and promotes hunting, generating small, localized trajectories. In the exploration state, the animal promotes locomotion and suppresses hunting, generating long-ranging trajectories that enhance spatial dispersion. We uncover a dorsal raphe subpopulation with persistent activity that robustly encodes the exploitation state. The exploitation-state-encoding neurons, together with a multimodal trigger network that is associated with state transitions, form a stochastically activated nonlinear dynamical system. The activity of this oscillatory network correlates with a global retuning of sensorimotor transformations during foraging that leads to marked changes in both the motivation to hunt for prey and the accuracy of motor sequences during hunting. This work reveals an important hidden variable that shapes the temporal structure of motivation and decision-making.

Call the Eckols: Present and Future Potential Cancer Therapies.

In recent years, an increased interest in marine macroalgae bioactive compounds has been recorded due to their benefits to human health and welfare. Several of their bioactivities have been demonstrated, such as anti-inflammatory, antioxidant, anticarcinogenic, antibacterial and antiviral behavior. However, there still lacks a clear definition regarding how these compounds exert their bioactive properties. Of all the bioactive compounds derived from marine macroalgae, attention has been focused on phenolic compounds, specifically in phlorotannins, due to their potential for biomedical applications. Phlorotannins are a diverse and wide group of phenolic compounds, with several structural variations based on the monomer phloroglucinol. Among the diverse phlorotannin structures, the eckol-family of phlorotannins demonstrates remarkable bioactivity, notably their anti-tumoral properties. However, the molecular mechanisms by which this activity is achieved remain elusive and sparse. This review focuses on the described molecular mechanisms of anti-tumoral effects by the eckol family of compounds and the future prospects of these molecules for potential application in oncology therapies.

Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish.

Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

Clusterdv: a simple density-based clustering method that is robust, general and automatic.

MOTIVATION: How to partition a dataset into a set of distinct clusters is a ubiquitous and challenging problem. The fact that data vary widely in features such as cluster shape, cluster number, density distribution, background noise, outliers and degree of overlap, makes it difficult to find a single algorithm that can be broadly applied. One recent method, clusterdp, based on search of density peaks, can be applied successfully to cluster many kinds of data, but it is not fully automatic, and fails on some simple data distributions. RESULTS: We propose an alternative approach, clusterdv, which estimates density dips between points, and allows robust determination of cluster number and distribution across a wide range of data, without any manual parameter adjustment. We show that this method is able to solve a range of synthetic and experimental datasets, where the underlying structure is known, and identifies consistent and meaningful clusters in new behavioral data. AVAILABILITY AND IMPLEMENTATION: The clusterdv is implemented in Matlab. Its source code, together with example datasets are available on: https://github.com/jcbmarques/clusterdv. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Synthesis and biological activity of a potent optically pure autoinducer-2 quorum sensing agonist.

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.

Impacts of S-metolachlor and terbuthylazine in fatty acid and carbohydrate composition of the benthic clam Scrobicularia plana.

S-metolachlor (SMOC) and terbuthylazine (TBA) are herbicides that commonly appear as active ingredients (a.i.) in the composition of plant protection products. In a previous work, experimental bioassays were performed using those chemicals to find suitable molecular biomarkers to assess its toxicity to the non-target species Scrobicularia plana. The results obtained showed that the pollutants produce mortality and biochemical changes at the species, namely in protein contents and enzymatic activity levels. Thus, for a better understanding of the total biochemical impacts of those pollutants in S. plana, the composition of fatty acids (FA) and carbohydrates (CH) of the survival organisms are investigated here. In addition, since this species is edible its biochemical profile is directly related to its nutritious quality, which is analysed in this study. Furthermore, the analyses were performed in two types of tissue - the muscle and visceral mass of each survival organism. The greatest changes in FA composition are observable in small size class, being the most sensitive size class both at the toxicological and biochemical level. FA contents are higher in small organisms, both at the field and under laboratory conditions, being the disparity between size classes higher in visceral masses than in muscles. Indeed, muscles adequately represent the FA profile since those molecules appear in higher content in this tissue compared to visceral masses, becoming the better indicator tissue of biochemical changes. Besides, using muscles, less amount of biomass is needed, so it turns out to be the most cost-effective tissue to be used as endpoint in future studies. FA profiles observed at SMOC and TBA exposure are different, organisms from TBA exposure presenting a lower nutritious quality, in terms of FA abundance and diversity, than the organisms exposed to SMOC. Still, SMOC produces reductions of HUFA, essential fatty acids that cannot be synthesized by the species. Moreover, HUFA (mostly EPA and DHA) occupied the greatest part of the FA composition of organisms exposed to the control treatments and to TBA; however, the decreases of HUFA caused by the SMOC exposure change the profiles and make SFA the most dominant group. These findings represent a risk of low occurrence of essential fatty acids in entire aquatic environments exposed to the chemicals studied. Regarding CH, glucose is the only monosaccharide found in S. plana which was expected since glycogen is the main polysaccharide in animal tissues. In general, the glucose content increases with a concentration of pollutants, whereas the glycogen concentration decreases, suggesting that the glucose is being released as a response to chemical stress. Thus, this work presents tools to assess biochemical impacts of S-metolachlor and terbuthylazine in aquatic systems and to goes deeper in the knowledge of these pollutants' toxicity to non-target species to predict its propagation through aquatic trophic webs.

Effects of a herbicide and copper mixture on the quality of marine plankton.

Pesticides and metals are often used in agriculture and are therefore often simultaneously discharged to nearby estuarine and marine areas. The effects of this organic-inorganic chemical mixture on food quality of aquatic organisms are currently unknown. In this study we test if a mixture of copper (inorganic) and the herbicide Primextra® Gold TZ (organic) affects the quality of the diatom Thalassiosira weissflogii and the copepod Acartia tonsa - two key species that fuel the local food-web. We quantified quality (i.e. energy content as food for the next trophic level) in terms of fatty acids, proteins and thiobarbituric acid reacting substances. We found non-additive effects (positive and negative) of the metal-herbicide mixture on the diatom and copepod species. In general, nutritionally important biochemical parameters of Acartia tonsa were most sensitive to the chemical stressors.

LsrF, a coenzyme A-dependent thiolase, catalyzes the terminal step in processing the quorum sensing signal autoinducer-2.

The quorum sensing signal autoinducer-2 (AI-2) regulates important bacterial behaviors, including biofilm formation and the production of virulence factors. Some bacteria, such as Escherichia coli, can quench the AI-2 signal produced by a variety of species present in the environment, and thus can influence AI-2-dependent bacterial behaviors. This process involves uptake of AI-2 via the Lsr transporter, followed by phosphorylation and consequent intracellular sequestration. Here we determine the metabolic fate of intracellular AI-2 by characterizing LsrF, the terminal protein in the Lsr AI-2 processing pathway. We identify the substrates of LsrF as 3-hydroxy-2,4-pentadione-5-phosphate (P-HPD, an isomer of AI-2-phosphate) and coenzyme A, determine the crystal structure of an LsrF catalytic mutant bound to P-HPD, and identify the reaction products. We show that LsrF catalyzes the transfer of an acetyl group from P-HPD to coenzyme A yielding dihydroxyacetone phosphate and acetyl-CoA, two key central metabolites. We further propose that LsrF, despite strong structural homology to aldolases, acts as a thiolase, an activity previously undescribed for this family of enzymes. With this work, we have fully characterized the biological pathway for AI-2 processing in E. coli, a pathway that can be used to quench AI-2 and control quorum-sensing-regulated bacterial behaviors.

Patchy sediment contamination scenario and the habitat selection by an estuarine mudsnail.

Since mudsnails are able to avoid contaminated sediment and that the contaminants in sediment are not uniformly distributed, the mudsnail Peringia ulvae was exposed to cadmium (Cd) spiked sediment and assessed for avoidance response in a heterogeneous contamination scenario. Four Cd concentrations were prepared and disposed in patches on dishes, which were divided in 25 fields (six fields for each sediment concentration); 24 organisms were deployed in the central field, with no sediment. Observations were made at 2, 4 and 6 h (corresponding to immediate response), 8, 10 and 12 h (very short term), and 24 h (short term). A trend to avoid contaminated patches was observed in the immediate and very short term. After 24 h exposure, the organisms exposed to the highest level of contamination seemed to have lost the ability to move and avoid contaminated patches. In a contamination scenario in which non- and contaminated sediment patches are heterogeneously distributed, local mudsnail populations can simply rearrange their locality without needing to move to a different habitat. Such less contaminated patches can become donor areas in a future recolonization scenario.

Spatial foraging segregation by close neighbours in a wide-ranging seabird.

Breeding seabirds are central-place foragers and therefore exploit food resources most intensively nearer their colonies. When nesting aggregations are close to one another density-dependent competition is likely to be high, potentially promoting foraging segregation (i.e. neighbouring colonies may segregate to search for food in different areas). However, little is known about spatial segregation in foraging behaviour between closely adjacent colonies, particularly in species that are wide-ranging foragers. Here, we tested for foraging segregation between two sub-colonies of a wide-ranging seabird, Cory's shearwater Calonectris borealis, separated by only 2 km, on a small Island in the North Atlantic. During the 2010 chick-rearing period, 43 breeding adults of both sexes were simultaneously sampled at both sub-colonies. A GPS logger was deployed on each individual and removed after several foraging trips at sea. Blood samples (plasma and red blood cells) were collected from each tracked individual for stable isotope analysis. Results indicated partial spatial segregation between the two sub-colonies during local foraging trips (i.e. those of ≤1 day duration and 216 km from the colony) accounting for 84.2% of all trips recorded. The location of the breeding sub-colony influenced the direction of travel of birds during local trips resulting in sub-colony-specific foraging areas. Although the oceanographic conditions associated with the foraging range of the two sub-colonies differed, no differences were found in the habitat exploited and in their estimated diets. This suggests that birds concentrated their feeding activity in patches of similar habitat and prey during the chick-rearing period.

Uncovering multiscale structure in the variability of larval zebrafish navigation.

Animals chain movements into long-lived motor strategies, exhibiting variability across scales that reflects the interplay between internal states and environmental cues. To reveal structure in such variability, we build Markov models of movement sequences that bridges across time scales and enables a quantitative comparison of behavioral phenotypes among individuals. Applied to larval zebrafish responding to diverse sensory cues, we uncover a hierarchy of long-lived motor strategies, dominated by changes in orientation distinguishing cruising versus wandering strategies. Environmental cues induce preferences along these modes at the population level: while fish cruise in the light, they wander in response to aversive stimuli, or in search for appetitive prey. As our method encodes the behavioral dynamics of each individual fish in the transitions among coarse-grained motor strategies, we use it to uncover a hierarchical structure in the phenotypic variability that reflects exploration-exploitation trade-offs. Across a wide range of sensory cues, a major source of variation among fish is driven by prior and/or immediate exposure to prey that induces exploitation phenotypes. A large degree of variability that is not explained by environmental cues unravels motivational states that override the sensory context to induce contrasting exploration-exploitation phenotypes. Altogether, by extracting the timescales of motor strategies deployed during navigation, our approach exposes structure among individuals and reveals internal states tuned by prior experience.

Uncovering multiscale structure in the variability of larval zebrafish navigation.

Animals chain movements into long-lived motor strategies, resulting in variability that ultimately reflects the interplay between internal states and environmental cues. To reveal structure in such variability, we build models that bridges across time scales that enable a quantitative comparison of behavioral phenotypes among individuals. Applied to larval zebrafish exposed to diverse sensory cues, we uncover a hierarchy of long-lived motor strategies, dominated by changes in orientation distinguishing cruising and wandering strategies. Environmental cues induce preferences along these modes at the population level: while fish cruise in the light, they wander in response to aversive (dark) stimuli or in search for prey. Our method enables us to encode the behavioral dynamics of each individual fish in the transitions among coarse-grained motor strategies. By doing so, we uncover a hierarchical structure to the phenotypic variability that corresponds to exploration-exploitation trade-offs. Within a wide range of sensory cues, a major source of variation among fish is driven by prior and immediate exposure to prey that induces exploitation phenotypes. However, a large degree of variability is unexplained by environmental cues, pointing to hidden states that override the sensory context to induce contrasting exploration-exploitation phenotypes. Altogether, our approach extracts the timescales of motor strategies deployed during navigation, exposing undiscovered structure among individuals and pointing to internal states tuned by prior experience.

The adaptor protein 2 (AP2) complex modulates habituation and behavioral selection across multiple pathways and time windows.

Animals constantly integrate sensory information with prior experience to select behavioral responses appropriate to the current situation. Genetic factors supporting this behavioral flexibility are often disrupted in neuropsychiatric conditions, such as the autism-linked ap2s1 gene which supports acoustically evoked habituation learning. ap2s1 encodes an AP2 endocytosis adaptor complex subunit, although its behavioral mechanisms and importance have been unclear. Here, we show that multiple AP2 subunits regulate acoustically evoked behavior selection and habituation learning in zebrafish. Furthermore, ap2s1 biases escape behavior choice in sensory modality-specific manners, and broadly regulates action selection across sensory contexts. We demonstrate that the AP2 complex functions acutely in the nervous system to modulate acoustically evoked habituation, suggesting several spatially and/or temporally distinct mechanisms through which AP2 regulates escape behavior selection and performance. Altogether, we show the AP2 complex coordinates action selection across diverse contexts, providing a vertebrate model for ap2s1's role in human conditions including autism spectrum disorder.

Phosphoenolpyruvate phosphotransferase system regulates detection and processing of the quorum sensing signal autoinducer-2.

Autoinducer-2 (AI-2) a signal produced by a range of phylogenetically distant microorganisms, enables inter-species cell-cell communication and regulates many bacterial phenotypes. Certain bacteria can interfere with AI-2-regulated behaviours of neighbouring species by internalizing AI-2 using the Lsr transport system (encoded by the lsr operon). AI-2 imported by the Lsr is phosphorylated by the LsrK kinase and AI-2-phosphate is the inducer of the lsr operon. Here we show that in Escherichia coli the phosphoenolpyruvate phosphotransferase system (PTS) is required for Lsr activation and is essential for AI-2 internalization. Although the phosphorylation state of Enzyme I of PTS is important for this regulation, LsrK is necessary for the phosphorylation of AI-2, indicating that AI-2 is not phosphorylated by PTS. Our results suggest that AI-2 internalization is initiated by a PTS-dependent mechanism, which provides sufficient intracellular AI-2 to relieve repression of the lsr operon and, thus induce depletion of AI-2 from the extracellular environment. The fact that AI-2 internalization is not only controlled by the community-dependent accumulation of AI-2, but also depends on the phosphorylation state of PTS suggests that E. coli can integrate information on the availability of substrates with external communal information to control quorum sensing and its interference.

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein.

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.

Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli.

Bacteria coordinate population-dependent behaviors such as virulence by intra- and inter-species communication (quorum sensing). Autoinducer-2 (AI-2) regulates inter-species quorum sensing. AI-2 derives from the spontaneous cyclisation of linear (S)-4,5-dihydroxypentanedione (DPD) into two isomeric forms in dynamic equilibrium. Different species of bacteria have different classes of AI-2 receptors (LsrB and LuxP) which bind to different cyclic forms. In the present work, DPD analogs with a new stereocenter at C-5 (4,5-dihydroxyhexanediones (DHDs)) have been synthesized and their biological activity tested in two bacteria. (4S,5R)-DHD is a synergistic agonist in Escherichia coli (which contains the LsrB receptor), while it is an agonist in Vibrio harveyi (LuxP), displaying the strongest agonistic activity reported so far (EC(50)=0.65µM) in this organism. Thus, modification at C-5 opens the way to novel methods to manipulate quorum sensing as a method for controlling bacteria.

Assessment of metal exposure (uranium and copper) in fatty acids and carbohydrate profiles of Calamoceras marsupus larvae (Trichoptera) and Alnus glutinosa leaf litter.

Physiological changes were explored in fatty acids (FA) and carbohydrate (CHO) composition in the shredder Calamoceras marsupus larvae (Trichoptera) and leaf litter (C. marsupus food) exposed to copper and uranium under natural and experimental conditions. We measured FA and CHO content in leaf litter and larvae specimens from reference and impacted streams, and exposed for 5 weeks to four realistic environmental concentrations of copper (35 µg L-1 and 70 µg L-1) and uranium (25 µg L-1 and 50 µg L-1). Regarding FA, (1) leaf litter had a reduced polyunsaturated FA (PUFA) content in metal treatments, s (14 to 33% of total FA), compared to natural conditions (≥39% of total FA). Leaf litter exposed to uranium also differed in saturated FA (SFA) composition, with lower values in natural conditions and higher values under low uranium concentrations. (2) C. marsupus had/showed low PUFA content under Cu and U exposure, particularly in high uranium concentrations. Detritivores also decreased in PUFA under exposure to both metals, particularly in high uranium concentrations. On the other hand, (1) microorganisms of the biofilm colonizing leaf litter differed in CHO composition between natural (impacted and reference) and experimental conditions, with glucose and galactose being consistently the most abundant sugars, found in different amounts under copper or uranium exposure; (2) CHO of detritivores showed similar high galactose and fucose concentrations in contaminated streams and high copper treatments, whereas low copper treatment showed distinct CHO profiles, with higher mannose, glucose, arabinose, and fucose concentrations. Our study provides evidence of metal exposure effects on FA and CHO contents at different trophic levels, which might alter the quality of food flow in trophic webs.

Marine macroalgae as a feasible and complete resource to address and promote Sustainable Development Goals (SDGs).

Because the world's population is increasing, science-based policies are needed to promote sustainable global development. It is important to maintain and restore the environment and help human society overcome the risks from industrialization and unsustainable exponential growth. In recent years, many studies have highlighted that macroalgae represent a key marine resource for ecological and sustainable living, thus helping to address today's global problems, such as water pollution, ocean acidification, and global warming. Macroalgae show the potential to provide innovative, ecofriendly, and nutritious food sources and natural compounds for various industries, such as biomedical, food, agricultural, and pharmaceutical industries. This review discusses how macroalgae can help us today and how they can promote a more sustainable way of life in the future. It also discusses the potential danger for ecosystems and the global population if these organisms are not part of the solution but part of the problem. Integr Environ Assess Manag 2022;18:1148-1161. © 2022 SETAC.

Are tolerance processes limiting the responses of Hediste diversicolor to cadmium exposure? A multimarker approach.

Cadmium (Cd) is considered a priority hazardous substance under the European Community Directive 2013/39 due to its ecotoxicity. The ragworm Hediste diversicolor (O.F. Müller, 1776), a common species in estuaries and coastal lagoons, plays an important ecological role in these ecosystems and is a suitable bioindicator of environmental chemical contamination. In this study, H. diversicolor was chosen as an ecotoxicological model with the aim of evaluating the responses to Cd contamination, considering a multi-biomarker approach (mortality, biometry, behaviour, Cd bioaccumulation, oxidative stress and damage, and energy metabolism). Also, the hypothesis of different tolerances resulting in different responses was evaluated, by collecting worms from three systems distinctly impacted by metal contamination (Mondego estuary, Óbidos Lagoon and Sado estuary - Portugal). Animals were exposed under laboratory conditions to cadmium (10, 50 and 100 µg/L), for 10 days. Significant differences were observed in responses amongst worms originating from the different sites. Organisms from the less impacted systems revealed greater effects on mortality, biomass decrease and burrowing behaviour, as well as higher bioaccumulation potential, after exposure to Cd. Biochemical and behaviour impairments were observed as a consequence of Cd exposure, although not in a concentration-dependant manner. The results obtained in this study reinforce the importance of integrating endpoint responses, at the individual and sub-individual levels, to assess potential changes induced by pollutants in the physiological status and fitness of H. diversicolor and help to predict what their ecological consequences might be.

Effects of wildfire ash on the growth and biochemical profiles of the aquatic macrophyte Lemna minor.

Wildfires are a social and environmental concern to the world due to their many adverse effects, including risk to the public health and security, economic damages in prevention and fight, ecosystems pollution, land usage sustainability, and biodiversity. In the Mediterranean region, these events have increased in the last years. Although several studies evaluated the impacts of the wildfires on the structure and function of the ecosystems and their communities, there is a lack of information at the biochemical level beyond the toxicological effects to the organisms. So, aiming to evaluate the potential toxic and biochemical effects of pine and eucalypt ash from high and low severity burned areas in the aquatic environments, L. minor growth, fatty acid and carbohydrate profiles were studied. Data showed that the wildfires ash from high severity burned areas are more toxic, with a higher growth inhibition than when exposed to ash from low severity burned areas. Considering the ash from low severity burned areas, eucalypt ash revealed to be more noxious to the macrophyte than pine ash. Furthermore, it was observed a decrease in the diversity and abundance of fatty acids content, comparing with the control. An opposite trend was observed in carbohydrates which increased with the organisms' exposure to almost all ash types, except in case of the organisms exposed to eucalypt ash from high severity burned areas, where carbohydrate content decreased.