France

Cold acclimation induces cardiac remodelling in rainbow trout. This remodeling can include changes in contractile function, cardiac hypertrophy as well as an increase in collagen content. To understand how collagen is regulated in the trout heart we have been studying the function of cultured trout cardiac fibroblasts, the cells responsible for collagen turnover. We have previously found that exposure of these cells to cyclical stretch and transforming growth factor beta 1 (TGF-β1) activate cellular signalling pathways involved in collagen deposition and increase collagen deposition. In the current study, we have examined the influence of different levels of cyclical stretch, testosterone, and angiotensin II (ANG II), on the cell signalling pathways that regulate collagen deposition. We also examined how low temperature and TGF-β1 affected these responses. We found that the effect of cyclical stretch on collagen related signalling pathways is frequency and magnitude dependent. In addition, testosterone treatment led to changes in gene expression that would support an increase in collagen deposition regardless of temperature. This includes changes in the expression of the gene transcripts for matrix metalloproteinase-9, collagen type 1 alpha, and tissue inhibitor of metalloproteinases 2. We also found that 1 µM of ANG II had no effect on collagen-associated gene expression. Our results also suggest that the effect of TGF-β1 on collagen deposition is temperature dependent.  The results of this work provide mechanistic insight into the regulation of collagen deposition in the trout heart and how this is affected by temperature change. This research is funded by NSERC. 

SEB Annual Conference 2022

The influence of environmental and endocrine factors on collagen regulatory pathways in rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts

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Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

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.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

The epidermis serves as a multifunctional interface between plants and their environment. 
The leaf surfaces of carnivorous plants such as the Nepenthes pitcher rim (peristome) are highly specialised. The structure of the peristome is unique, holding a hierarchical pattern of radial ridges, grooves, and overlapping steps, that render it highly wettable and slippery to insects when wet. Virtually nothing is known about how the peristome's characteristic microtopography forms inside the hollow, developing pitcher bud. We used SEM imaging to document this process for the first time. We found that a sequence of distinct epidermal patterning events (cell organisation, conical cell formation, papillate cell outgrowth, and cell elongation) governs peristome development. All of these individual processes are common and conserved in leaf epidermal patterning across plant lineages. We hypothesise that Nepenthes co-opted these processes for peristome development and combined them in a novel way. In order to probe the genetic underpinnings of the sequential epidermal patterning events, we identified external morphological markers on the growing pitcher bud and linked these to a timeline of internal peristome development. This will allow us to target specific stages of surface patterning with confidence, which is crucial for investigating gene expression patterns and gene function during development. 













Nepenthes pitcher plants recombine common patterning processes to create a complex hierarchical epidermis structure

Food waste is a global issue with 1/3 of all food lost or wasted annually. Tomato is a culturally important fruit that provides nutrition to a global market but is highly perishable and requires carefully controlled storage and transportation to maintain shelf life. Losses occur throughout the entire supply chain as often the symptoms of spoilage are detected too late for effective repurposing. There are existing methods that are used in a commercial environment for detecting spoilage, however, these methods are often slow, destructive, and expensive. It is important that a non-destructive, high-throughput and cheap technology becomes readily available that can used throughout the supply chain to detect spoilage biomarkers for effective repurposing of produce. This study investigates the use of attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy as a rapid, quantitative method for detecting decay in surface tissue of tomato fruit. Spectra was analysed using chemometric multivariate analysis of the fingerprint region (400 cm-1 to 1500 cm-1). In a time-series from harvest to spoilage, we identified differences between the time points in the series demonstrating the molecular changes in the fruit surface through the ripening and decaying period. Distinct chemical peaks were detected which represent potential biomarkers for spoilage and were associated with specific time points. Notable fruit properties associated with these peaks include the changes in levels of phenolic compounds, cell wall polysaccharide modifications including pectin and cellulose. ATR-FTIR provides a low-cost method for in vivo detection of spoilage biomarkers and biochemical changes. 

Effective detection of spoilage biomarkers in tomatoes 

Understanding the mechanisms determining these limits is, therefore, essential in our general understanding of thermal tolerance of ectotherms. It has been put forth that oxygen limits cardiac performance at high temperatures, although this has rarely been explicitly or thoroughly tested. It has also been suggested that hyperkalemia may negatively impact cardiac function under elevated temperatures. We hypothesized that low environmental oxygen and elevated blood [K+] would reduce the upper thermal limits of the heart in a marine teleost (Girella nigricans). Using the Arrhenius Breakpoint Temperature Test, we assessed fish (n=12 per treatment) under hyperoxic (200% air saturation), normoxic (100% air saturation), and hypoxic (20% air saturation) conditions under a range of plasma [K+]. We also measured ventricle lactate content and venous blood oxygen pressure (PO2) to determine if there were common critical points in these metrics driving cardiac collapse. We found significant differences in cardiac upper thermal limits (Arrhenius breakpoint temperature [TABT], peak maximum heart rate (peak fHmax], temperature of peak heart rate [Tpeak], and temperature at which the heart becomes arrhythmic [TARR]) in fish exposed to hypoxia during the trial compared to those exposed to normoxia or hyperoxia. We also found significantly higher ventricular lactate content in fish exposed to hypoxia. Elevated [K+] may be correlated with decreases in TABT, but there were no other significant correlations. These results suggest that oxygen limitation can drive cardiac collapse at high temperatures. 

Hypoxia lowers cardiac thermal limits in a marine teleost (Girella nigricans)

Many ectotherms can optimize their physiology during development to match current or future environments (developmental plasticity) and there is often variation in the capacity for plasticity between individuals and populations. However, the degree to which developmental stochasticity contributes to this variation is not well understood. This is important because developmental instability may limit an individual’s ability to match their physiology to the environment. Here, I cloned 18 genotypes of the model organism Nematostella vectensis (starlet sea anemone) to test variation in developmentally plastic phenotypes both within and between individuals. Specifically, I analyzed the repeatability of developmentally plastic phenotypes by developing three clones per genotype (N=18) at each of three developmental temperatures (13°C, 21°C, and 29°C); 162 clones total. I hypothesized that genotypes would vary in their capacity for plasticity and that more plastic genotypes would have greater developmental stability at the thermal extremes. Our results suggest that within-individual variation in developmental rate is low at 29°C and high at 13° and 21°C — especially relative to inter-individual variation. Our preliminary results also suggest that metabolic rate it is a more repeatable trait than developmental rate. This variation is interesting because it may represent limitations in regulatory processes during development or variation in ecological strategies to overcome environmental stress (e.g., diversified bet-hedging). Further analyses will leverage this dataset to test for potential costs or trade-offs associated with plasticity.

Developmental Responses to Thermal Stress in the Starlet Sea Anemone

Climate change is causing rapid warming of Arctic regions. The impact of temperature rise on the survival of Arctic marine species such as the keystone species polar cod (Boreogadus saida) depends on the potential acclimatory or evolutionary adjustments of thermal limits in their most sensitive life stages. According to literature data on acute thermal limits differences in thermal preferences suggest that reproducing adults are more vulnerable than non-reproducing adults or juveniles, however a cause-and-effect understanding is lacking. An in vivo approach of magnetic resonance imaging (MRI) was used to compare the physiological performance of unanaesthetised, reproducing and non-reproducing polar cod during acute warming. Individual fish were placed in the MR scanner that was continuously perfused with aerated seawater. Inside the MR scanner, 0°C acclimated polar cod were exposed to acute warming from 0°C to 8,5°C at a rate of 1.5 °C every two hours. A set of specialized MRI techniques was applied to measure physiological performance parameters such as heart rate and monitor the energy status at different temperatures. At the end of the experiment fish were sacrificed and blood and tissue samples were taken for further analyses.

Effects of acute warming on reproducing and non-reproducing polar cod (Boreogadus saida)

Mitochondrial function depends on direct interactions between respiratory proteins encoded by genes in two genomes, mitochondrial and nuclear, which evolve in very different ways. Serious incompatibilities between these genomes can have severe effects on development, fitness and viability. The effect of subtle mitonuclear mismatches has received less attention, especially when subject to mild physiological stress. We investigate how various stressors affect phenotypic traits, mitochondrial function, metabolic pathways and gene expression in Drosophila larvae, and adults of both sexes. Flies fed either a high protein diet, the glutathione precursor N-acetyl cysteine (NAC), or the NADH precursor nicotinamide riboside (NR) and had sex, life-stage, and genotype-specific responses to these stressors. Metabolomic results point to changes in TCA cycle flux, while respirometry analysis shows changes in substrate use and complex I function. Our results support the notion that subtle mitonuclear mismatches can lead to diverging responses to mild physiological stress, undermining fitness in some cases, but surprisingly improving outcomes in other mismatched fly lines.

Mitonuclear interactions impact responses to metabolic and redox stress at different life stages in Drosophila melanogaster

Birds and mammals are endotherms which maintain a high and constant body temperature. At rest, the average body temperature of birds is ~41°C and of mammals ~37°C. However, during a high-intensity exercise (flying/running), muscle temperature can reach 44°C and 42°C respectively. At the mitochondria level the fuel selection differs between the two clades. While mammals display a general dependence on carbohydrate, birds can use both carbohydrates and lipids oxidation to respond to energy demands. Many studies have highlighted this last point but without considering the muscular temperature at different metabolic intensities as a combined factor with the preferential use of available substrate for ATP synthesis. Herein, we compared isolated mitochondria from skeletal muscle of pigeons and rats of similar body mass and measured the oxidation rates at 3 assay temperatures (37°C, 40°C, 43°C). We used 2 mitochondrial substrates: a combination based on carbohydrate (pyruvate + malate) and based on lipid (palmitoyl-L-carnitine + malate). We found that isolated rat mitochondria exhibited higher oxygen consumption with carbohydrate-based energy substrates compared to metabolism with lipid-based substrates, a difference that was exacerbated at high temperature. The ability of avian mitochondria to oxidize different substrates in equivalent way, even at high temperatures would trigger a more flexible response to challenging environments and the high energy requirements of flight.  

Mitochondrial fuel selection in birds and mammals: a question of muscular temperature

Eusocial insects, such as colonies of ants, wasps, and bees, operate as an integrated collective with tasks allocated among individuals. There is a long tradition of detailed work on the female workers that carry out colony tasks. Males, however, are typically ignored, because they do not perform work, and/or they simply disperse from the natal nest. In the Western honey bee, (Apis mellifera), males (drones) live in the colony throughout their lives, but prior research focuses almost exclusively on their behavior outside of the nest (mating flights), while ignoring their in-nest behavior. To understand the in-nest behavior of drones across their entire lives, we used the BeesBook tracking system to track 192 individually-marked drones at 3 fps for 25 days. We used these trajectories to extract behavioral parameters, including how drones developed, how they moved, and where they spent their time. While drones do spend around 21 hours of their time immobile at the periphery of the nest, as expected, we also found that drones have periods of in-nest hyperactivity, in these times moving faster than even workers. This hyperactivity develops in drones after 7 days, occurs daily between 14:00 and 17:00, and is synchronized across the cohort. While active, drones move towards the nest entrance, and gaps in tag detections indicate trips outside of the nest (presumably flying to mating sites). Surprisingly, we even found this behavior on days of poor weather, when drones would not fly, suggesting that drones must inspect the daily weather themselves. While drones are typically attributed to being the lazy members of the colony, here we show that they have hyperactive bouts that surpass even worker activity. The duration, however, is short, likely to conserve energy. Drones are part of the colony’s organization, even if their role is simply to become active once daily to maximize opportunities for mating success. 

Hyperactive and synchronized pre-mating behavior of honey bee drones inside the nest

Understanding the metabolic costs of locomotion is key for understanding the energy budgets of wild animals, yet can be challenging to quantify, especially for aquatic animals that may be difficult to observe or recapture. Here we quantified relationships between tail-beat frequency and swimming speed in free-swimming fishes at a large pelagic display aquarium at the Nordsøen Oceanarium, Denmark. Using stereocamera footage of free-swimming fish, we were able to determine the swimming-speed and body size of individuals from four study species: Atlantic mackerel, horse mackerel, needlefish, and the starry smooth-hound shark. While there were strong relationship between tailbeat frequency and swimming speed, the steepness of this relationship varied among species and between swimming types. Furthermore, for Atlantic mackerel, the relationship between swimming speed and tailbeat observed in free-swimming individuals differed from that measured for individuals swimming within a swim tunnel. This may be due to differences in how momentum can be used to maintain speed while freely swimming, or the energetic saving that can be gained by swimming in schools, and suggests caution be used when applying relationships between oxygen uptake and swimming speed derived from swimming tunnel respirometry to free-swimming fishes. Overall, more work examining the swimming behaviour and bioenergetics of free-swimming fishes is required to understand energy use in differing environments in the wild.

Relationships between tail-beat frequency and swimming speed in free-swimming fishes

The mTORC1 pathway is believed to play a very important role in the regulation of de-novo protein synthesis across vertebrates as well as many other organisms, although the evidence in fish is presently limited. This pathway can be specifically inhibited by rapamycin, which stops the up-regulation of protein synthesis while allowing constitutive protein synthesis to continue. This contrasts to the classically used cycloheximide, whose mechanism is now understood to cause total protein turnover inhibition. Since it is de-novo protein synthesis that is thought to underly the post prandial increase in metabolism (systemic dynamic action or SDA) and of course growth itself, this method has the potential to promote both a better understanding of protein metabolism but also to vastly improve feed development. We tested this system in Perca flvitalis using a protocol with fasted fish that where either force-fed 3% of body mass or sham fed with saline. Using an intra-peritoneal injection of 20mg/kg Rapamycin the post prandial increase in protein synthesis was completely inhibited resulting in fish that had the same rate of protein synthesis as sham fed fish. Oral administration of Rapamycin in feed was also attempted but failed to inhibit post prandial increases in protein synthesis, suggesting that oral dosing requires elevated doses. This study indicates that Rapamycin and mTORC1 regulated protein synthesis in fish has great potential for increasing our understanding of fish growth.

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