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Anthropogenic carbon dioxide emissions have been increasing rapidly in recent years, driving pH and oxygen levels to record low concentrations in the oceans. Eastern boundary upwelling systems such as the California Current experience exacerbated ocean acidification and hypoxia (OAH) due to the physical and chemical properties of the transported deeper waters. Research efforts have significantly increased in recent years to investigate the deleterious effects of climate change on marine species, but have not focused on the impacts of simultaneous environmental stressors. Additionally, few have explored the physiological impacts of these environmental stressors on early life stages, which are more vulnerable and represent natural population bottlenecks in organismal life cycles. The physiological response of the red sea urchin (Mesocentrotus franciscanus) to co-occurring stressors was assessed by exposing larvae to varying OAH conditions observed along the California coast. Graded responses, proportional to treatment severity, indicate that minor shifts in OAH affect morphology and gene expression, especially related to growth and pH homeostasis. In addition to this gradient, breakpoints can be detected, representing drastic shifts in response curves and a potential environmental threshold below which growth and performance are profoundly impacted. The phenotypic and transcriptional breakpoints occurred between DO of 2.64-3.67 mg/L and pH 7.6-7.85 with an additional transcriptional breakpoint between DO 0.88-1.54 mg/L and pH 7.2-7.45. By assessing the physiological response and gene expression of these species, we can inform future management actions to mitigate OAH-related impacts and maintain fishery populations.

SEB Conference Prague 2024

Transcriptional and Morphological Response of Red Sea Urchin Larvae (Mesocentrotus franciscanus) to Combined Ocean acidification and Hypoxia

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SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

In recent years, Nephrotic Syndrome (NS) has emerged as a significant challenge in global public health. NS encompasses a spectrum of diseases affecting kidney function, with Membranous Glomerulonephritis (MGN) being the most prevalent in mammals.Common indicators of NS include protein loss in urine and decreased blood albumin levels. Primary glomerulonephritis is the leading cause, with Anti-Phospholipase A2 receptor antibodies (anti-PLA2R) serving as a diagnostic marker for MGN, as per Kidney Disease: Improving Global Outcomes (KDIGO) guidelines.
Our study employed various machine learning techniques to develop predictive models using disease classification data and examination results such as complete blood count, blood and urine biochemistry. After data refinement, the dataset was split into training and testing sets, with Python utilized to build machine learning models. Statistical analysis revealed significant differences (p<0.05) in BUN, Creatinine, and eGFR between normal individuals and those predicted to have the disease. The machine learning process incorporated Synthetic Minority Oversampling Technique (SMOTE) to address imbalanced data and 5-Fold Cross-Validation for validation. Final models utilized the Random Forest algorithm and eXtreme Gradient Boosting (XG Boost), achieving an Accuracy and AUC of 0.83, indicating robust predictive performance.
Our methodology, leveraging routine blood and urine tests alongside machine learning algorithms, enables accurate prediction of MGN. In the future, this methodology holds promise for application in mouse research and potentially diagnosing human diseases in clinical.

Predicting Membranous glomerulonephritis in Mammals Using Machine Learning

After prolonged noise exposure, we lose our hearing ability, which is often temporary and typically recovers (resilience). Previously, we established the desert locust, Schistocerca gregaria, as a model for auditory recovery after noise exposure. In this work we wished to track the rate of short-term recovery after noise exposure. In order to do this, we conducted over 600 independent measurements of auditory system function after noise exposure and quantified both the transduction current from individual auditory neurons and spiking responses from the auditory nerve. For the sound-evoked nerve response we find a rapid increase in recovery over the first 8 hours, followed by a more gradual recovery over the next 20 hours. For the transduction current we find a more steady recovery after noise exposure. We wanted to understand the genetic basis of this recovery. Therefore, we extracted RNA from auditory Müller’s organ to identify transcriptomic changes responsible.







Previously, we established the desert locust, Schistocerca gregaria, as a model for auditory robustness and resilience. We found differences in resilience when comparing young and aged locusts. Regardless of age, the sound-evoked auditory nerve response recovers completely in young and aged locusts, however, the sound-evoked transduction current does not recover in aged locusts. We thus identify a potential plastic mechanism that compensates for a noise-induced decrease in transduction current in aged locusts. We then examine the auditory resilience of the locust ear in higher resolution, at the level of both the sound-evoked auditory nerve response and transcriptomic changes over the first 28 hours after noise exposure. We find a rapid increase in recovery over the first 8 hours following noise exposure, followed by a more gradual recovery over the following 20 hours. Our high-resolution transcriptomic results offer insight into the fundamental mechanisms of auditory recovery. 




Tracking the rate of recovery of the locust's auditory system after noise-exposure.

Insects exchange respiratory gases through a gas-filled, highly branched tracheal system, that extends from paired segmental sipracles to fine tracheoles. Gas exchange between the tracheoles and the tissues is entirely diffusive, but locusts, and many other insects, rely on convection of gas within the tracheal system for facilitating gas transport from the environment to the tracheoles even at rest. In this study we compared structural and functional tracheal system traits between migratory locust (Locusta migratoria) populations originating in the Tibetan Plateau (TP; 4000 above sea level) and in low altitude Hainan Province (HP), China. We hypothesized that the TP locusts have adapted to low environmental oxygen availability in their natural habitat. Following multiple generation rearing at sea-level laboratory conditions TP locusts were significantly smaller than HP conspecifics, yet their tracheal volume was significantly higher. Mass-specific metabolic rates were similar in both populations, but gradual exposure to decreasing environmental oxygen levels yielded a more pronounced metabolic (and ventilatory) response in non-adapted HP compared with TP. Critical oxygen partial pressures, under which metabolic rates cannot be maintained, were marginally lower in TP compared with HP (1.8±0.2 and 2.4±0.2 kPa, respectively), but just short of statistical significance (p=0.07). Nevertheless, this evolved variation in respiratory system function may be more pronounced during activity, when tissue oxygen demand is higher. Moreover, exposure to hypoxic conditions throughout development resulted in lower adult body mass in HP, but not TP locusts, compared with development in normoxia, highlighting long-term effects of evolved respiratory adaptation.

Respiratory adaptation to high altitude in migratory locusts

The Asia-Pacific aquaculture, renowned for its success, faces mounting climate change
challenges. Intensified heatwaves and erratic weather disrupt operations, with summer
temperature extremes breaching optimal thresholds. This leads to a proliferation of pathogenic
microorganisms and harmful substances, jeopardizing the industry's sustainability and output.
Resveratrol (RSV), a natural polyphenolic compound, is well-known for its antioxidant properties
and modulates inflammatory responses against pathogens. Our study aims to assess the efficacy
of RSV-supplemented diets in combating bacterial infections and mitigating inflammation in
aquaculture species during summer conditions.
Our study uses tropical tilapia (Oreochromis mossambicus) to examine the impact of RSVsupplemented
diets on gut microbial composition. PacBio 16s rRNA sequencing unveiled a
decrease in potentially pathogenic bacteria (Aeromonas) and enhanced in beneficial probiotics
(Cetobacterium) in both the gut and gills of tilapia fed with RSV. Moreover, in vitro investigations
demonstrated that RSV inhibits Aeromonas growth. Further analysis using RNA-Seq to investigate
the microbial composition changes induced by RSV. We found that RSV as a dietary supplement
can reduce the expression levels of immune response-related genes (RAGE, IL-1β, IL-6, cox2).
Overall, our findings suggest that RSV supplementation holds potential as a promising
dietary supplement for tropical fish, aiding their resilience against the adverse effects of summer
temperature spikes induced by climate change and contributing to the sustainability and
productivity of the aquaculture industry.

Enhancing Tropical Fish Resilience to Heat Stress: Exploring the Protective Role of Resveratrol as a Dietary Supplement

Climate change has emerged as a critical issue in recent years. Previous research has highlighted the increase in prevalence of Vibrio in marine environments due to rising sea surface temperatures. This rise in pathogenic Vibrio has led to increased infection rates among humans and aquatic organisms. In freshwater ecosystems, Aeromonas stands out as the predominant pathogen. Both Vibrio and Aeromonas are known for their ability to thrive in diverse aquatic environments, posing significant health risks to both aquatic life and human populations. Therefore, understanding the dynamics of these pathogens in response to environmental changes is crucial for developing effective strategies to mitigate their impact on public health and ecosystem.
Our study primarily focuses on investigating Aeromonas, a facultative anaerobic Gram-negative bacillus commonly found in freshwater and wastewater environments. Aeromonas poses significant health risks to freshwater fish and can also lead to diseases in human, including diarrhea, wound infections, skin infections, pneumonia, and sepsis. Multiple Aeromonas isolates were gathered and subjected to strain typing via Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) techniques. Furthermore, we explore the antibiotic resistance profiles of Aeromonas isolates to understand the dynamics of resistance development. Bacteriophages were screened from various freshwater sources. Once corresponding phages were identified, they were purified for further investigations. 
These investigations offer potential applications in aquaculture to improve fish survival rates or in medical contexts to mitigate the pathogenic effects of Aeromonas on human disease.

Phage-Based Eradication of Aeromonas: A Novel Approach against Aquatic and Clinical Pathogens

The gills of farmed Atlantic salmon are susceptible to damage from various stressors, including toxins released by harmful algae (HA). Microcystin is a HA-toxin that affects fish and typically induces toxicity through oxidative stress with detoxification mainly occurring in the liver by glutathione (GSH). While the effect of microcystin on liver is well studied, its impact on gills, which are the first point of contact with microcystin has been overlooked. In this study, we explored the toxic effects of microcystin-LR (MC-LR) on the Atlantic salmon gill epithelial cell line (ASG-10) and whether N-acetyl-l-cysteine (NAC), a precursor of GSH, decreases these potential impacts.
The ASG-10 cells were exposed to different concentrations of MC-LR (0, 5, 10, 20, 40 µg/ml) in presence or absence of NAC (1 mM). Cell viability, reactive oxygen species (ROS) production, GSH level, and cell migratory behaviour/wound healing, were measured at different time points for up to 5 days post-exposure.
Cell death was only significantly increased after treatment with 40 µg/ml MC-LR after 5 days. There was a significant negative impact of MC-LR on proliferative and migratory ability of cells and the gap in the cell monolayer did not close when treated with 20 and 40 µg/ml of MC-LR. Interestingly, MC-LR had no impact on ROS production and GSH level in ASG-10 cells. However, NAC treatment was associated with reduced ROS and increased intracellular GSH as expected. These data indicate that MC-LR has negative effects on cell proliferation and may impair gill tissue healing and regeneration following damage.


The effect of harmful algal toxin on the Atlantic salmon gill epithelial cell line (ASG-10)

Natural vibrations are used by animals to communicate, to sense predators, detect prey, forage and reproduce. Vibrational communication assists both invertebrates and vertebrates to retrieve information from their surrounding environment. However, anthropogenic activities that are in direct contact with the seabed (such as pile-driving and mining) produce both sound in the water column and vibrations in the seabed. Exposure to vibrational noise has the potential to harm bottom-dwelling species in a similar way to water borne noise, potentially eliciting behavioural or physiological changes, or even causing physical damage. Research regarding substrate vibrations and marine invertebrates is in its infancy, with the vibrational sensitivities of most marine species relatively unknown; there is therefore a pressing need to understand the use of vibrations, since their usage defines how animals interact with their environment, as well as how human activity might interfere with natural biological processes. Here, we outline our project plan which will utilise different experimental setups to investigate the sensitivities of marine invertebrates to substrate-borne vibration. Preliminary studies will be done under controlled laboratory conditions to compare vibrational sensitivities, between and within taxa, through repeatable and reproducible methodologies. Overall, we aim to understand the effects of vibrational noise on key marine invertebrates and highlight the need for a shift in underwater noise research, to recognise vibrational noise in addition to water-borne sound.

Establishing methodologies for testing the sensitivity of marine invertebrates to substrate-borne vibrations

Microplastics (MPs), defined as plastic particles smaller than 5 mm in size, are considered a global environmental and human health problem, due to their ubiquity and persistence in the aquatic ecosystems. Considering the problematic of MPs, the present study aimed to assess the toxicological effects of MPs and copper (Cu) in zebrafish brain. Polyethylene (PE) and polystyrene (PS) MPs were selected because of their extensive commercial use and high accumulation in aquatic environments.
For this purpose, zebrafish were exposed for 21 days to Cu (25μg/L), PE and PS (1mg/L) and their mixtures (PE-Cu and PS-Cu). After 21 days, fish behavior was assessed, followed by their euthanasia with an overdose of buffered tricaine methanesulfonate, and excision of the whole brain for the biomarker’s evaluation. The biochemical parameters related to oxidative stress [Reactive oxygen species, lipid peroxidation]; antioxidant activity [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized states (GSSG) of glutathione levels]; and detoxification (glutathione-S-transferase) were assessed.
Data showed that the exploratory/locomotor, anxiety-related, and social behaviors were not significantly affected by Cu, PE, PS, and the respective mixtures. Considering the biochemical parameters, the GPx activity was significantly decreased in PE-Cu, in relation to the control group.
Considering our findings, the long-term behavioral and biochemical alterations in response to heavy metals and MPs, and its implications in fish population fitness still need to be clarified.

This work is supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020.

The effect of polyethylene and polystyrene microplastics combined with copper in behavior and biochemical biomarkers of zebrafish (Danio rerio) brain

Several snake species sport horn-like appendages over their snouts and/or eyes. Although these structures have recently attracted scientific attention, their functional significance remains unknown. One hypothesis holds that the cephalic appendages may work as heat radiators/dissipators, keeping temperature stable during sudden changes in heat load. Following this hypothesis, we expect that horns heat and cool at different rates than the rest of the head and have a stabilizing influence on brain temperature. To test this idea, we used water-filled 3D-printed head models of vipers, the snake family with the highest number and diversity in horned species. After validating our method, we compared the heating and cooling of an array of hornless and horned models in a temperature-controlled environment and monitored horn and head temperatures. Our preliminary results show that horns indeed heat and cool at different rates than the rest of the head. However, the brain region of models with and without horns heated and cooled at similar rates. Therefore, unless they are accompanied by a special blood vessel cooling and countercurrent exchange system, the horn-like structures of snakes probably cannot function to regulate head/brain temperature. We are currently investigating the possibility of such rete mirabile in snakes, using micro-CT scanning and by monitoring heating and cooling behaviour of snakes in-vivo.

Thermal properties of horns in vipers - A preliminary but innovative study using 3D-printed models

While microplastics (MPs) have become the focus of an increasing amount of research in the last decade, there is still limited knowledge on the effects these pollutants have on the physiology of aquatic organisms. The aim of this study was to determine how exposure to polyethylene (PE) microplastics (38-42 µm, 250µg/L) impacts the physiology of two representative fish species; zebrafish (Danio rerio) and killifish (Fundulus heteroclitus) under different conditions. Behavioral changes and ammonia excretion rates were compared between zebrafish exposed to MPs and controls. Killifish were examined during acclimation to changing salinity with and without the added stress of MPs exposure. Zebrafish exposed to MPs showed a significant increase in ammonia excretion rates (p=0.042) compared with controls. Behavioral changes due to MPs exposure varied, showing significant differences in surfacing (p=0.021) and hyperventilation (p=0.036) behaviors. During salinity acclimation of killifish, MPs exposure eliminated the normal increase in drinking rates commonly seen in euryhaline fishes moving into higher salinity. This type of effect would have a significant impact on the capacity of killifish to rapidly acclimate to changing salinity. 


Transcriptional and Morphological Response of Red Sea Urchin Larvae (Mesocentrotus franciscanus) to Combined Ocean acidification and Hypoxia

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Predicting Membranous glomerulonephritis in Mammals Using Machine Learning

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