Czechia

While legally protected in Portugal since 1988, the Iberian wolf still faces conservation problems, mainly due to habitat fragmentation, caused by human activity, and poaching.
This study is part of a wolf monitoring project that aims to establish the reference situation of the wolf population to assess the impact of a mining exploration in northern Portugal. We measured faecal glucocorticoid metabolite concentrations in wolf scats as a non-invasive method to determine stress levels, using an enzyme immunoassay kit.
Wolf scats were collected in the area of two wolf packs, Barroso and Leiranco. Molecular methods were used to confirm the species, assign sex, wolf pack, and, whenever possible, individuals. During two years, we identified 15 individuals (11 males and 4 females) from Barroso’s pack area and 10 individuals (7 males and 3 females) from Leiranco’s pack area, of which we analysed 133 and 60 samples, respectively.
Results show that the Leiranco pack, inhabiting the area with higher human density, has higher levels of faecal cortisol. We also found, for both wolf packs, a significant seasonal effect; higher levels were found during the autumn, which corresponds to the main hunting season.
The present work is the first developed in Portugal aiming the characterization of cortisol levels in free-ranging wolves. Since this work is included in a long-term project, increasing the number of collected samples, and the analysis of other relevant variables influencing cortisol levels, will improve our understanding of the factors affecting wolf populations.

SEB Conference Prague 2024

Non-invasive assessment of stress hormone cortisol in two wild packs of Iberian Wolf (Canis lupus signatus) in northern Portugal

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Chemical cues play a crucial role in aquatic environments, with most research focusing on cues related to injury and stress. However, there have been limited studies on identifying specific chemical components within these cues, unveiling a knowledge gap in chemosensory biology. Our previous studies have demonstrated that heat stress can likewise prompt the release of olfactory cues, that propagate within a group and induce stress in naïve developing zebrafish (Danio rerio). Using metabolomics, we identified glucose, phosphocholine, and 3-mercaptolactate as potential components of heat-induced stress metabolites. Hatching was impaired by stress metabolites. In this study, we verified whether 3-mercaptolactate and phosphocholine are indeed potential stress metabolites by assessing their impacts on hatching rate and gene expression in control metabolite (CM) and thermal stress metabolite (SM) groups. Indeed, both compounds significantly impaired hatching and as a result were found to cause bent tails in zebrafish larvae. Genes associated with the metabolism of these compounds (prg4a -phosphocholine; ldha and mpst – 3-mercaptolactate) were differentially expressed, with the expression of mpst, associated with the metabolism of 3-mercaptolactate, trending downwards in metabolite receivers. While further research into the mechanism of action is ongoing, these results indicate how lipid and sulfur-containing compounds can be used for heat-induced stress communication in developing embryos.

Phosphocholine and 3-mercaptolactate as potential stress metabolites in zebrafish (Danio rerio)

Fjords are physically isolated areas characterised by distinct oceanic conditions,
making them perfect case-
studies to investigate the consequences of local adaptation and in situ long-term
acclimatisation in marine
organisms. In these environments, global change drivers such as ocean acidification
(OA) pose a significant
threat to marine ecosystems, including keystone zooplankton species such as the
copepod Calanus glacialis.
Both local adaptation and long-term acclimatisation may lead to differential responses
of C. glacialis to OA
along its range of geographical distribution. Consequently, our study aimed to explore
how these phenomena
can influence the ability of various populations of C. glacialis to cope with future OA
conditions. To do so,
copepods were collected from four fjords spanning from southern Norway to Svalbard,
and exposed in the
laboratory under controlled conditions to seven different increasing concentrations of
pCO 2 (300 to 30,000
ppm) over time to explore the threshold at which each population shows signs of
sensitivity to OA. Targeted
metabolomes for each treatment were profiled using high-performance LC-MS/MS, to
quantify 49 metabolites
involved in metabolism. Our analyses will help identifying signs of local
acclimatisation, which would be visible
if copepods show population-specific metabolome profiles in response to altered ocean
chemistry. Moreover,
breakpoint analyses will be used to characterise potential abrupt changes in the
relationships between the
pCO 2 stress gradient and copepods’ metabolomics response. Our work will provide
crucial insights to help
predicting the long-term impacts of OA on C. glacialis populations.

Consequences of local adaptation/in situ acclimatisation of an Arctic copepod on its metabolomics response to an ocean acidification gradient

General biotic and abiotic contamination of water bodies is widely recognised. Zebra mussel (ZM) is commonly used in biomonitoring of water quality for its capacity to filter a great volume of water, and accumulate contaminants present in aquatic environments. The ZM’s digestive gland is a key organ in assessing the health status of the organism and concentrates high amounts of naturally occurring mixtures and pollutants. ZMs’ organs can adequately present the aquatic environment's health status and “early warning” for biomonitoring. The gut microbiota may significantly influence health and responses to environmental stressors of the organisms. The interaction between sentinel species and their microbiota has prompted questions, especially regarding their potential as indicators in environmental monitoring. Nevertheless, environmental factors can impact the composition and metabolism of gut microbiota responsible for breaking down and detoxifying xenobiotics.
To advance our understanding of microbiota's role, we conducted both field and controlled studies. The first aim assesses the gut microbiota composition (16s rDNA sequencing) and physiological state in ZMs in-situ. The organisms were caged for 21 days in oligotrophic to eutrophic sites varying in food sources, bacterial load and water chemistry. In parallel, controlled experiments aimed to determine the optimal conditions (food quantity) for maximizing the filtration rate and effective removal of microorganisms. It occurred with the initial algae concentration of 10^5cells/mL and microbial populations at 10^5UFC/mL. Higher filtration rates cause higher contact with contaminants and feeding. This first step will enable us to investigate the impact of stressors under laboratory conditions on gut microbiota.


How Does the Gut Microbiota of Zebra Mussels (Dreissena polymorpha) Serve as a Key Indicator of Organism and Aquatic Health?

Mussels from the Mytilus edulis species complex are euryhaline osmoconformers that adjust their internal osmolarity to match that of their surroundings. This adaptive strategy poses a significant challenge to the metabolic machinery of the mussels, including mitochondria. An earlier study showed that mussels from different populations achieve optimal mitochondrial respiration at salinities near their prevailing habitat salinity. Given the euryhaline biology of mussels, we hypothesised that osmotic optima for the mitochondrial function will show plasticity to salinity acclimation. To test this hypothesis, mussels from the southern Baltic Sea (Mytilus edulis x trossulus transition zone) were acclimated to either 6 (hypoosmotic) or 15 (control) salinity and the mitochondrial performance curve was generated by measuring respiration of isolated hepatopancreas mitochondria at a range of osmotic concentrations (230–915 mOsm/kg). We found that the optima of the oxidative phosphorylation (OXPHOS, indicative of ATP synthesis capacity) and mitochondrial coupling were shifted towards a lower osmolality under hypoosmotic conditions. Additionally, the OXPHOS respiration was consistently higher, while efflux of reactive oxygen species (ROS) and fractional electron leak were lower in mussels acclimated to salinity 6 compared to those at salinity 15. This response of mitochondria from hypoosmotically exposed mussels resembles that of mitochondria from north Baltic Sea mussels (M. trossulus) that were collected and acclimated at their natural salinity (6) in a previous study. Thus, it appears as though a salinity of 6 offers optimal conditions for mussel populations from the north and south Baltic Sea.

Salinity dependent mitochondrial respiration in mussels of the Mytilus edulis species complex

The Antarctic Ocean is one of the only places on earth where all biological processes happen between -2 and 2°C. At these temperatures, our models for life fall apart. Diffusion, protein folding, energy and metabolism are all processes that are poorly understood in the cold, where processes are slow and energy is scarce. The gap in our understanding of cold-adapted life comes partly from the lack of suitable equipment to properly observe these systems. Our team has recently developed a method to perform high resolution imaging of cold live samples, therefore shedding a new light to observe these systems in physiological conditions. The development of this new method offers perspectives for studying the rate of molecular, cellular and sub-cellular processes. Observing cells from Antarctic animals in vivo reveals some surprising dynamics, in particular when comparing the speed of organelle movement in the cold compared to temperate organelle speed. This opens avenues to investigate how the energy budget of cold-adapted cells is spent, and what survival strategies have evolved from such a constrained environment.
This new microscopy method could also be used to study the dynamics of similar systems when their environment is warming up, or for other applications in biotechnology, including but not limited to questions about phase separation, diffusion models and protein aggregation.


Microscopes and Antarctic fish: How developing high-resolution microscopy for 0°C imaging sheds a new light on cold-adapted systems.

Climatic changes involved an important increase in the global mean surface temperature and frequency of heat waves, being a significant health risk reported in diverse big and medium-sized mammals. The analysis of energy expenditure components is key for understanding the processes of heat loss and thermoregulation. One of the highest heat expenditure activities is feeding; mammal metabolism can increase around 25-50% of the resting metabolic rate (RMR). It has been suggested that smaller animals could exhibit elevated fitness under warmer temperatures; however, this has not been tested on small wild mammals during the postprandial state. We addressed this issue in two non-endangered wild mouse species, the wood mouse (Apodemus sylvaticus) and the Algerian mouse (Mus spretus) kept in laboratory conditions. RMR and postprandial heat increment (HI) were measured by open flow respirometry at cool conditions (20°C) and within the thermoneutral zone (28°C). RMR was measured after overnight fasting of 16 -21 hours. Food was provided at 10% of the animal's body mass to evaluate the HI. We expected that mice would prioritize thermoregulation efficiency to food consumption. At warmer temperatures mice decrease their food intake to reduce HI, allowing efficient thermoregulation without compromising RMR. These adjustments could evidence possible changes in diet selection at higher temperatures related to the nutritional composition to reducing thermogenesis.

DOES THE TEMPERATURE INCREMENT AFFECT THE POST-PRANDIAL ENERGY METABOLIC RATE IN SMALL WILD MAMMALS?

The effect of temperature on biological functions can vary among individuals of the same species according to the environment they inhabit. In this sense, intraspecific variations in the energetic balance can provide insights on microevolutionary processes occurring at the population scale.
The aim of our work was to investigate intraspecific differences in fundamental energetic functions, namely energy acquisition via feeding, energy expenditure via standard metabolic rate, and energy allocation via growth in response to temperature, in a species living along a large latitudinal gradient. We exposed the common periwinkle, Littorina littorea, from 10 different locations in North America, to 12 temperatures (5 to 27 C) in a common garden experiment, and measured survival, feeding rate, growth, metabolic rate, and CTmax. Populations’ thermal performance curves were obtained and suggested that the thermal energy budget differs among different locations. Energy acquisition through feeding and energy allocation to growth differed only for locations at the species limits, which may be subjected to stronger selective pressure. Survival and CTmax were found to be lower at higher temperatures for snails from lower latitudes, this likely reflecting increased energetic needs for maintenance. However, the remarkable differences in the shape of metabolic rate may reflect local variations in energy expenditure strategies. Specifically, snails from different climates may use different strategies to adjust their energy budget to changing temperatures. Our study provides evidence of potential local and clinal adaptation of the investigated species to varying environmental conditions, helping to define its current (and likely future) range limits.


Variations in the energetic balance among populations of a marine gastropod living along a large latitudinal gradient.

Sediment runoff is a primary driver of coral reef degradation globally. Herbivorous fish (i.e., those that eat algae) are seen as a critical defense by managers worldwide to control the overgrowth of algae on corals. Yet, populations of these herbivores are rapidly declining on nearshore reefs impacted by sedimentation. Current evidence firmly establishes that fish gills undergo structural changes in response to increased levels of suspended sediment (SuS), suggesting a link between morphological alterations and potential respiratory impairment. Direct measurements of oxygen uptake in fish during SuS exposure would provide valuable insight into the physiological mechanism underpinning fish abundance. For this study, we 3D-printed novel respirometry chambers, explicitly designed with a cone-bottom shape, to allow sedimented water to enter underneath test fish and keep sediment suspended throughout respirometry experiments. Here, we present preliminary results on the oxygen uptake rate in the bioculturally important Pacific bullethead parrotfish, Chlorurus spilurus, measured not only post-exposure but also concurrently during exposure to SuS. We hypothesized that fish exposed to SuS would have a reduced metabolic scope and an extended recovery time to return to their standard metabolic rate—resulting in higher excess post-exercise oxygen consumption and signifying substantially reduced gill function. 



Metabolic responses to suspended sediment in a Hawaiian herbivorous reef fish

Vortical interactions permeate the natural world, from trout navigating streams to flying insects. These interactions serve to reduce the energy expenditure required for transportation while simultaneously enhancing thrust and lift. This study delves into the specific phenomenon known as wake capture, in which organisms harness the vortices shed from their own movements to extract or utilize energy. From jellyfish maneuvering through water to fruit flies navigating the air, wake recapture proves instrumental in minimizing energy expenditure. Through the analysis of three distinct case studies, we illuminate the significance of wake capture across various mediums: water, air, and at the interface. By comprehending the underlying principles and utilizing dimensionless numbers, a universal framework can be established to deepen our understanding of efficient locomotion across different environments.

Vortical interactions in nature

Warmer water temperatures and heat waves exacerbated by climate change are affecting the physiology and thermal tolerance of aquatic ectotherms, including fishes. Warming waters also alter infection dynamics with many species of parasites predicted to increase in prevalence and intensity as temperatures rise. Since parasites can alter host physiology, infection may have detrimental effects on host thermal tolerance. Ectotherms can increase their thermal tolerance through acclimation. Yet, the exposure time required for fishes to fully acclimate remains understudied. Furthermore, no studies have explored the interaction between parasite infection and acclimation time. We tested the effect of acclimation time on three pumpkinseed sunfish (Lepomis gibbosus) populations from lakes in Québec, Canada that differ in the prevalence and intensity of infection with helminth parasites. Pumpkinseeds were acclimated at either ambient (22°C) or +5°C (27°C) warm treatment for between 3h and 60 days and then tested in a critical thermal maximum (CTmax) protocol. CTmax in pumpkinseeds held at ambient temperature was highest in the uninfected lake. CTmax increased with acclimation time in all three lakes, but the speed of increase was slower in the two lakes with high parasite prevalence. However, these differences were not driven by individual infection intensity. Our findings indicate that prolonged acclimation time can influence the thermal tolerance of pumpkinseeds. Our study highlights the importance of considering different populations when studying a physiological trait and using adequate exposure time to fully acclimate the fishes.

Non-invasive assessment of stress hormone cortisol in two wild packs of Iberian Wolf (Canis lupus signatus) in northern Portugal

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Phosphocholine and 3-mercaptolactate as potential stress metabolites in zebrafish (Danio rerio)

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