Belgium

Excretion of nitrogenous waste is essential for all animals, and the type of waste is largely determined by water availability. Adult teleostean fish excrete ammonia primarily via their gills, which adds to water pollution in intensive aquaculture, and can impact fish health. Larval fish initially produce urea and shift to ammonia excretion after hatching, first via the skin, then via the gills. 
We studied the transition from cutaneous to branchial ammonia excretion. We investigated the expression and localization of ammonia transporters (rhesus glycoproteins) in zebrafish up to 8 days post-fertilization (dpf) and coupled these data to measurements of nitrogenous waste excretion and expression of marker genes for osmoregulation and urea production. We discovered a differential change in rhesus glycoprotein localization: Rhag and Rhbg translocate to gill filaments before 5 dpf, while Rhcgb translocates to the gills between 5 and 6 dpf and is localized in ionocytes. These findings indicate two pathways of ammonia excretion that transition independently, which is corroborated by correlating gene expression and developmental stage: post-hatch larvae up to 6 dpf show increased rhag, rhbg and rhcga expression, while 7-8 dpf zebrafish show increased expression of rhcgb and osmoregulatory marker Na+ /H+ -exchanger 3b. Expression of the uncharacterized rhcgl1 gene increased in a similar pattern, hinting towards involvement of additional rhesus glycoproteins in ammonia excretion. Our research suggests two distinct ammonia excretion pathways in zebrafish, and their relative contributions to ammonia excretion, as well as effects of environmental factors (e.g., pH and environmental ammonia) are subject of our future studies.

SEB Conference 2025

How zebrafish handle their nitrogenous waste: Determining the skin-to-gill transition of rhesus glycoproteins

ammonia transporters

immunohistochemistry

ammonia

development

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SEB Conference 2024

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Deforestation has drastically reduced natural roost sites for European bats, with artificial roost boxes being a common conservation measure. However, concerns arise regarding the potential overheating of artificial roosts, particularly during summer heat waves. Female bats roosting in summer maternity colonies are especially vulnerable due to the hotter and more humid conditions compared to solitary males. This study investigated sex and seasonal differences in heat tolerance and evaporative cooling capacity in four species of vespertillinoid bats. Using indirect calorimetry and thermometry, we measured thermoregulation at air temperatures (Ta ) ranging from 28 to 48°C during spring (male bats only, females in reproductive phase) and summer (both males and females). Contrary to predictions, there were no sex differences in maximum Ta  tolerated or evaporative cooling efficiencies. While there were also no seasonal differences in maximum Ta  tolerated, male bats unexpectedly had lower maximum cooling efficiencies during summer compared to spring. Moreover, there were sex and seasonal differences in the patterns of increasing evaporative heat loss (EHL) with increasing Ta . Female bats started increasing EHL at higher Ta  than males during summer, and male bats increased EHL more gradually with rising Ta  in summer compared to spring. These differences in heat-dissipation strategies suggest potential adaptations for enhanced water conservation under hotter and more humid conditions, at both sex and seasonal levels. Using roost temperature and EHL data, we estimated daytime evaporative water costs of bats using artificial roosts, revealing that small bats are already at risk of lethal dehydration during present-day heat waves.

Seasonal and sex differences in the thermoregulatory responses of European vespertilionid bats at high air temperatures 

Temperature is a key environmental constraint on insect population size and distribution, and a crucial factor limiting establishment and spread of invasive species. For most insects, cold exposure results in progressive loss of ion balance dependent on both exposure duration and intensity. This ionoregulatory collapse, characterized by extracellular hyperkalemia, causes cell death, tissue damage and eventually death. However, unlike these chill-susceptible species, recent work shows that truly cold hardy insects (e.g. freeze-avoidant mountain pine beetle larvae) only partially lose ion balance during severe cold exposure and maintain extracellular [K+ ] just below lethal cellular limits. Whether this is a common capacity amongst cold-hardy species is unknown, as are the mechanisms behind this phenomenon. Asian longhorn beetle larvae (Anoplophora glabripennis; ALB) are a destructive invasive forest pest that accumulates cryoprotectants and becomes partially freeze-tolerant in winter. We tested whether cryoprotectants can increase cold tolerance by acting as osmoprotectants that mitigate cold-induced ionoregulatory collapse. Specifically, we explored the ability of this species to maintain ion balance in the cold by measuring hemolymph ion concentrations (e.g. Na+ /K+ ) and cryoprotectants during a prolonged cold stress. ALB larvae lab-reared on artificial diet were sampled at multiple time points during cold exposure at temperatures approaching and beyond their freezing point to measure hemolymph osmolality and ion and cryoprotectant concentrations. Results from this study suggest that ALB have an ability to prevent ionoregulatory collapse in the cold, and by extension that such an ability may be a pre-requisite to evolving freeze tolerance or freeze avoidance in insects.

Keep an ion it! Prevention of cold-induced ionoregulatory collapse in the freeze-tolerant Asian longhorn beetle (Anoplophora glabripennis)

Most insects, including Drosophila melanogaster, suffer from a progressive loss of ion and water balance that causes cell, and ultimately organismal, death. This ionoregulatory collapse has been linked to temperature-induced reductions in membrane-bound transporter activity (e.g. Na+ /K+ -ATPase, H+ -ATPase) in the renal system. Cold acclimation can improve low temperature tolerance and is associated with an increase in water and ion transport rates in the Malpighian tubules (analogous to kidneys) of cold acclimated flies, but a paradoxical reduction in the activity of ATPases in the same tissues. This raises questions about whether the current models of ion transport apply to cold-acclimated insects, or if modulation by other mechanisms, such as membrane structural reorganization allow for improved renal function in the cold. Here, we report the results of transcriptomic sequencing of the anterior Malpighian tubules of warm and cold-acclimated flies before, during, and after a cold stress. We used these results to construct gene networks, using a weighted gene correlation network analysis method. These results link networks of genes to specific traits and suggest that physiological state of tubules before a cold stress is more important to preventing downstream injury than a short-term response to chilling. They further suggest that cold acclimation elicits a tissue repair response in tubules during recovery, as opposed to an immune response seen in warm acclimated flies. Together, these findings allow us to generate hypotheses on how cold acclimation allows insects to adjust their cold tolerance.

Networking in the cold: connecting renal transcriptional plasticity to chill injury prevention through gene network analysis.

The disease vector, Aedes aegypti, can better survive low temperature exposure following cold acclimation. This plasticity may be contributing to this species’ recent range expansion. However, the molecular mechanisms behind this ability remain unexplored. MicroRNAs (miRNAs) can drive rapid changes in gene expression and are known to regulate thermal plasticity in other animals. We used a bioinformatic approach to identify three novel miRNAs in Ae. aegypti. After acclimating adult female Ae. aegypti to cold (15°C) and warm (25°C) conditions, we used RT-qPCR to perform differential expression analysis on these novel miRNAs and four other miRNAs downregulated during diapause in another mosquito species. Two novel miRNAs, aae-miR-novel2 and aae-miR-novel3, were upregulated in cold-acclimated individuals. Two described miRNAs associated with lipid metabolism and reproduction, aae-miR-8-3p and aae-miR-13b-3p, were downregulated in the cold-acclimated group, suggesting these processes are affected during cold acclimation. Temperature alone can alter miRNA target genes, and the thermodynamic parameters used to predict miRNA targets are typically established assuming body temperatures relevant to endotherms, rather than those applying to poikilothermic ectotherms. Here, we instead tested for cold-influenced targets of miRNAs of interest, predicted to only become thermodynamically stable at 15°C. This approach provides insight into the putative roles that these novel miRNAs could play in cold acclimation. Many predicted targets were involved in processes associated with insect low temperature survival, like ion homeostasis and membrane stability, while others suggest roles for understudied processes. Our results suggest that miRNAs play a regulatory role in mosquito cold acclimation that deserves further attention.

Thermal profiling of novel and known miRNAs during cold acclimation in Aedes aegypti.

Emily Lechner1* , Mafalda Tomas1 , Leon Pfeufer1 , and Fredrik Jutfelt1 
1  Department of Biological and Environmental Sciences, Faculty of Science, University of Gothenburg, 41390, Gothenburg, Sweden
* Author for correspondence: emily.lechner@bioenv.gu.se
A warming climate can have consequences on the performance of ectotherms through increases in metabolic demand. Understanding how variation in water oxygen saturation and supra-optimal temperatures affect the vulnerability of fishes to heatwaves and hypoxic zones is imperative to predicting anthropogenic impacts on wild organisms. However, we do not yet have a full understanding of how water oxygen saturation influences aerobic capacity, and how this may be temperature dependent.
This study investigates the constraints imposed by warming and hypoxia on aerobic performance in two model species with distinct thermal niches: the cold-adapted rainbow trout (Oncorhynchus mykiss) and the warm-adapted zebrafish (Danio rerio). Here, we present swim performance and aerobic scope profiles of these two species in response to variable oxygen saturation and temperature regimes. We used racetrack swim-tunnel respirometry to measure swim performance and maximum metabolic rates along a gradient of air saturation (from 25% to 200%). Fish will be acclimated to three temperatures that are ecologically relevant to their species (sub-optimal, optimal, supra-optimal). The results will be discussed in relation to the mechanisms limiting aerobic performance in fish and will address gaps in knowledge that relate to oxygen dynamics and thermal resilience of aquatic ectotherms. This work is essential for improving projections of aquatic biodiversity under future climatic scenarios.

Exploring Fish Aerobic Scope in a Changing Climate 

Marine heatwaves (MHWs) are becoming more frequent, intense, and prolonged with ongoing global changes. This study aimed to evaluate the impacts of sex-specific exposure to MHWs on the reproduction and energy metabolism of the amphipod Gammarus locusta. Females (F) and males (M) were sampled at a coastal lagoon and kept individually during a 7-day period under a control (20 °C) or a MHW (25 °C) scenario. After 7 days, females and males were sampled for mitochondrial and digestive enzymes activities, while the remaining specimens were used to form breeding pairs in different combinations (FControl /MControl , FControl /MMHW , FMHW /MControl , and FMHW /MMHW ). These breeding pairs were kept at control temperature (20 °C) until either reproducing or dying. Survival, reproductive success, fecundity and offspring body-sizes were subsequently determined. Reproductive success and fecundity were extremely reduced when both adults were exposed to MHWs, when compared to breeding pairs with both adults coming from the control treatment. Significant differences were also observed in the sizes of neonates, with offspring from control couples displaying smaller sizes, when compared to those from breeding pairs in which one of the adults was exposed to MHWs. We predict that MHWs will induce sex-specific enzymatic responses, such as differential energy allocation via changes in mitochondrial enzyme activity and the disruption of the endocrine system-related enzymes. MHWs imposed significant metabolic challenges on G. locusta, leading to sex-specific responses. Our findings highlight the need to consider sex as a biological variable in global change research to better predict species resilience and population stability.

When the Heat Rises: Sex-Specific Effects of Marine Heatwaves on Reproductive Success and Energy Metabolism in the Amphipod Gammarus locusta

In a changing climate, organisms are experiencing increasing thermal stochasticity. However, the role of the unpredictability of conditions on the biological consequences of thermal variability remains poorly explored. In particular, the sensitivity of metabolic performance to temperature could have large consequences from individual fitness to ecological communities via its influence of resource intake. In this context, we investigated the effect of ecologically relevant patterns of stochastic and cyclic thermal variations on the energetic budget of a pelagic freshwater fish, the European chub (Squalius cephalus). Fish (n=131) were exposed to constant temperatures regimes (13°C, 19°C or 25°C) or experienced profiles with variable temperatures sharing the same mean (19°C) and variance. Variable groups underwent either stochastic variations randomly oscillating between 13°C and 25°C, or cyclic variations following a circadian pattern. Both energy expenditure through whole-organism oxygen consumption and energy intake with predation rate were assessed on the same fish after one and six weeks of exposure to thermal treatments. After one week of exposure, preliminary results show that metabolic responses are driven by the mean temperature experienced, conversely to predation rates that are even sensitive to the pattern of variations. These results point out the necessity of accounting for thermal variability in experimental contexts and shed the light on our upcoming results investigating the plasticity of responses after a prolonged exposure. Additionally, such findings raise questions about a potential shift of thermal optima across biological traits and therefore highlight the relevance of assessing multiscale effects of environmental variability in studying climate change.

Does predictability matter? Effect of stochastic and cyclic thermal variations on energetic budget in a freshwater fish

Coastal habitats are among the most productive ecosystems on Earth, acting as biodiversity hotspots and essential nursery grounds for marine life. In Scandinavia, seagrass Zostera marina habitats provide food and shelter for many fishes. However, these meadows are typically confined to shallow areas due to water turbidity and eutrophication. As global warming intensifies the severity, frequency, and duration of heat waves, eelgrass-associated fishes are increasingly at risk of thermal stress. Yet, the extent of species-specific thermal limits and their plasticity for acclimation remains poorly understood. To address this gap, we investigated the effects of warming on wild eelgrass-associated fish during summer, when their thermal history is highest and temperature extremes are most impactful. We exposed twelve species from six families to a control temperature (19°C) or a simulated heatwave (23°C) for five days. We then studied their thermal tolerance using the Critical Thermal Maximum (CTmax) test and measured the temperature at loss of equilibrium. Our results showed differences in upper thermal limits across species, partly influenced by phylogenetic history. Most species exhibited an acclimation response to warming, reflected by an increase in CTmax, though some species acclimated faster than others. Our findings emphasize the importance of considering species-specific thermal responses when assessing the ecological impacts of heatwaves on coastal fish communities.

Capacity for rapid thermal acclimation differs across eelgrass-associated fishes

Pacific salmonids are anadromous and generally migrate to the Bering Sea in summer to forage. Chum salmon (Oncorhynchus keta), one of the most abundant Pacific salmonids in the summer Bering Sea, spends a significant portion of its life at sea. However, due to its migratory nature, little is known about its foraging migration ecology. This study investigated the open-ocean migratory behavior of chum salmon using satellite pop-up archival tags (PSATs). A chum salmon, captured in the central Bering Sea (56.0°N, 175.0°W) on 30th July 2024, was tagged with a PSAT (miniPAT-348) and released. The PSAT surfaced as scheduled 75 days later in the North Pacific Ocean near the waters off Amchitka Island, Aleutians (50.8°N, 179.3°E). Over the next seven days, the PSAT transmitted depth, water temperature, and activity data, with 50.4% of the recorded data successfully retrieved. From 60 days after release until three days before surfacing, the PSAT recorded sea surface temperatures above 20 °C, suggesting predation by an endothermic fish. The estimated migration path, excluding the predation period, indicated a generally southward movement from the release site but remained in the Bering Sea. The salmon primarily used the surface (above 20 m) but occasionally dived below the thermocline (below 40 m). The fish did not exhibit diel vertical movement; however, it tended to increased activity around sunrise and sunset. This study provides insights for the open-ocean migratory behaviour of chum salmon but further study is required to explore this in more detail.

Ocean Migration Behaviour of Chum Salmon in the Summer Bering Sea Using Pop-up Satellite Archival Tag　

Environmental temperature is a key determinant of fitness in insects, influencing survival, reproduction, and behaviour. With climate change increasing the frequency and intensity of extreme thermal events, it is crucial to understand the adaptational capacity of insects to this new thermal reality. While the impacts of acute thermal stress on physiological traits are well-documented, less is known about the developmental effects of such extreme events, particularly regarding environmental buffering mechanisms like parental care behaviours. In this study, we investigated how exposure to a heatwave during the larval stage ofNicrophorus vespilloidesinfluenced reproductive success and parental care in adulthood. Larvae were exposed to either a heatwave (26°C for 72 hours) or kept at control conditions (20°C) and assessed for reproductive and behavioural outcomes in the adult focal individuals and their offspring. We found that heatwave exposure significantly reduced survival to eclosion in the focal larvae. However, there was no significant impact of the heatwave on either the parental behaviour or the duration of care exhibited. Similarly, no developmental effects of thermal stress were observed on adult reproductive traits or for offspring fitness. These results suggest that extreme thermal events early in life do not necessarily have long-term detrimental effects, contrary to recent findings on ontogenetic sensitivity in insects. This highlights the complex interactions between temperature and insects’ life history and underscores the need for further research to more accurately assess resilience in a rapidly changing world.

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Seasonal and sex differences in the thermoregulatory responses of European vespertilionid bats at high air temperatures

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