France

Testing how evolutionary cold adaptation affects metabolism and performance of fish we compared the acute responses to warming of the stenothermal Antarctic eelpout Pachycara brachycephalum and of the eurythermal common eelpout Zoarces viviparus from the North Sea. We investigated whole animal oxygen consumption rate and ammonia excretion. As excretion of ammonia via the gills reflects whole animal metabolic patterns, especially nitrogen and amino acid metabolism, we also investigated the temperature dependent protein synthesis rate of gills in vivo and connected it to respiration and ammonia excretion. 
Both species were exposed to temperature changes within their species-specific thermal window:  P. brachycephalum -acclimated to 0°C- at a rate of 2°C day-1 until 10°C, and Z. viviparus - acclimated to 4°C - at a rate of 3°C day-1 until 22°C. Once temperature stabilized (within 12 hours), oxygen consumption rates were measured repeatedly and water samples were taken to determine the ammonia excretion of each fish individually. In a parallel experiment, we investigated the temperature dependent protein synthesis rate (Ks) of gills after intraperitoneal injections of 13C-labeled phenylalanine. The gill tissue was sampled 1.5 and 3 hours after the injection.
All measured parameters followed an exponential increase with warming for both species, however, with different Q10 values (e.g. Q10 protein synthesis rate P. brachycephalum 4.2 vs. 2.8 Z. viviparus). Protein synthesis rates at the same temperatures were similar in both species (P. brachycephalum vs. Z. viviparus: 4°C Ks= 2.5 vs. 2.6 % day-1; 10°C Ks= 4.5 vs. 4.3% day-1) indicating no cold compensation regarding protein synthesis in gills of the Antarctic eelpout. It will be discussed how these results are related to oxygen uptake and ammonia excretion.

SEB Conference 2022

The impact of acute warming on the respiratory system of fish – a comparison of eelpouts from Antarctica and temperate zones

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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.

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 found 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.

Mitochondria are most known for their role in energy production, but they are also implicated in many other cellular functions (apoptosis, ROS production, cell proliferation, aging). In humans, the mitochondrial DNA was believed to encode only 37 genes involved in energy production and mitochondrial translation. However, recent studies have shown that within these genes could hide several small alternative genes (genes with translation initiation or termination sequences distinct from the reference gene sequences in which they are found) coding for micropeptides with a broad spectrum of functions that can be newly added to the large list of functions in which mitochondria are involved.

Following the discovery of the mitochondrial alternative gene mtaltnd4, we try to elucidate the functions of the gene and its peptide (MTALTND4) by studying: (1) the expression pattern of the peptide in different human tissues and blood plasma, (2) the involvement of the gene in the major mitochondrial functions by exposing cells to various treatments, (3) the interaction partners of MTALTND4 by mass spectrometry and (4) the impact of the peptide on cell transcriptome. 

We have found that mtaltnd4 may potentially produce a signaling protein in response to stress that would regulate certain cell metabolic processes and there is surely more to discover.











The alternative gene mtaltnd4: uncovering the mitochondria’s hidden functions

The progression of climate warming will expose ectotherms to transient heatwave events and temperatures above their tolerance range at increased frequencies. It is therefore pivotal that we understand species’ physiological limits and the capacity for various controls to plastically alter these thresholds. Exercise training could have beneficial impacts on organismal heat tolerance through improvements in cardio-respiratory capacity, but this remains unexplored. Using juvenile Chinook salmon (Oncorhynchus tshawytscha), we tested the hypothesis that exercise training improves heat tolerance through enhancements in oxygen-carrying capacity. Fish were trained once daily at 60% of their maximum sustainable swim speed for 60 min. Tolerance to acute warming was assessed following three weeks of exercise training, measured as the critical thermal maximum (CTMax). CTMax measurements were coupled with examinations of the oxygen carrying capacity (haematocrit, haemoglobin concentration, relative ventricle size, and relative splenic mass) as critical components of the oxygen transport cascade in fish. Contrary to our hypothesis, we found that exercise training did not raise the CTMax of juvenile Chinook salmon with a mean CTMax increase of just 0.35°C compared to unexercised control fish. Training also failed to improve the oxygen carrying capacity of fish. Exercise training remains a novel strategy against acute warming that requires substantial fine-tuning before it can be applied to the management of commercial and wild fishes. 

Does exercise training improve heat tolerance in Chinook salmon (Oncorhynchus tshawytscha)?

Analogous to the situation in several lepidopteran and dipteran insects also the larval stages of some echinoderms and hemichordates (ambulacraria superphylum) evolved highly alkaline midguts up to pH 10.5. Despite its pivotal role in species´ sensitivity to changes in seawater pH, the underlying epithelial transport mechanisms are largely unknown. Using ion-selective microelectrodes we found that pluteus larva of the purple sea urchin not only have highly alkaline midgut fluids (pH ~9) but also a substantial reduced sodium concentration (150 mM) compared to the surrounding sea water (450 mM). We pharmacologically investigated the role of Na+/H+ exchangers in intracellular pH (pHi) regulation and midgut proton and sodium maintenance using the NHE inhibitor 5 (n ethyl n isopropyl)amiloride (EIPA). While life cell imaging demonstrated the importance of NHEs in pHi regulation of midgut epithelia cells, basolateral EIPA application decreased midgut pH whereas luminal application, via micro-injections, increased midgut [Na+], without affecting pH. Gene expression analyses identified putative NHE candidates for midgut pH and Na+ homeostasis. Based on this information an antibody was generated against the sea urchin Slc9a2, which showed that the protein is localized in luminal membranes of the midgut. Additionally the use of specific vivo morpholino knock down of spslc9a2 provoked an increase in midgut [Na+] without affecting pH underlining the role of this transporter in midgut Na+ maintenance. 
This work provides new insights of NHEs being involved in ion regulatory mechanisms, especially the maintenance of pH and [Na+] in larval midgut fluids and thereby show conserved features to insect and vertebrate digestive systems, which may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.






Midgut Na+ and pH homeostasis is differentally regulated by Na+/H+ exchangers in the sea urchin larva

Otoliths are paired calcified structures and are used for hearing and keeping balance of teleost fishes. Otoliths are mainly composed by aragonite, one of the three calcium carbonate polymorphs. However, the otolith carbonate structures often transform from aragonite to another polymorph, vaterite and the mechanism for this transformation remains unclear. To examine the transformation mechanism, we established a new method for changing otolith carbonate polymorphs. Ten individuals of an inbred strain of Oryzias latipes were independently reared in aquaria with adding SrCl2・6H2O to tap water. The other set of individuals were reared in normal tap water as a control group. Sagittal otoliths were removed after 114¬¬–146 days of experimental period. To distinguish between aragonite and vaterite polymorphs, strontium (Sr) and magnesium (Mg) compositions of otoliths were analysed using an electron probe micro analyser. The individuals reared in Sr-rich water had otoliths of which the margins were replaced with vaterite, while control individuals had entirely aragonite otoliths. Our results clearly show that the addition of Sr to rearing water induces polymorphic transformation from aragonite to vaterite. Considering that the polymorphic transformation from inorganic calcite to inorganic aragonite is induced by adding Mg to water, thermodynamic mechanisms possibly affect the observed transformation. Our findings have a potential to explore the impact of having vaterite otolith on fishes as well as to improve understanding of vaterite-forming mechanisms.

A new method to induce polymorphic transformation from aragonite to vaterite in Oryzias otoliths

The adaptive significance of sequential hermaphroditism, typically found in tropical fish and marine invertebrates, has been successfully explained by the size-advantage principle (SAP) and notable exception has not been reported. Here we focus on a novel type of protandrous sequential hermaphroditism in an intertidal holothurian as a case not amenable to SAP. This species typically participates as a male in the first three spawning events, and then spawn eggs at the fourth, which is followed by the second sex change to release sperm again. By incorporating published empirical findings into evolutionary invasion analyses, we successfully specified the theoretical conditions that a population containing the reversible sex change is evolutionarily stable. Our scheme is a natural extension of the classical Fisher's principle of sex allocation, and is also capable of consistently explaining a strongly biased operational sex ratio found in sequential hermaphrodites.

Protandrous hermaphroditism with male regression: An explanation based on evolutionarily stable mixed strategies

The relationship between temperature and wingbeat frequency has been the focus of many studies over several decades and it has been shown repeatedly, across taxa, that warmer temperatures lead to higher flapping frequencies. This phenomenon is thought to be driven chiefly by the muscle properties of the flight motor. There are knock-on consequences for the kinematic pattern of the wing stroke that is required in order to generate aerodynamic forces sufficient to balance body weight. Specifically, we might predict that as frequency increases, the stroke amplitude or angle of attack must decrease to avoid producing excess force. 
Since mosquitoes have very high baseline wingbeat frequencies for their size, they operate with low wing stroke amplitudes, resulting in unusual aerodynamic mechanisms that place a greater burden of lift generation on the supinatory and pronatory rotations during the wingbeat cycle. Temperature-modulated increases in frequency reduce the stroke amplitude still further. Here, we investigate the aerodynamics of male Anopheles gambiae mosquitoes during flights recorded over a range of temperatures spanning almost 10 degrees Celsius. The kinematics were captured in a new multi-camera arena, and the flow fields were subsequently simulated by solving the incompressible Navier-Stokes equations. We present noteworthy variation in Anopheles kinematics with temperature and the mechanism of aerodynamic force production across the range of recorded kinematics.

The effect of temperature on mosquito aerodynamics

The rabbit is a widely used model system in comparative and clinical sciences for understanding correlations between anatomy and function in the masticatory system. Experimental and simulation studies have hypothesised that the rabbit is able to modulate chewing mechanics to process a variety of food types with high efficiency. Through synchronously measuring 3D jaw motions (using biplanar x-ray videography), muscle length change (using fluoromicrometry) and muscle activity (using electromyography) during feeding on foods of varying material properties and maximum bite-force in-situ experimentation we have begun to test these hypotheses. Processing tougher food types resulted in an increase in whole chew-cycle duration, and particularly in the slow-closing proportion of chewing. Additionally, working side muscles appear to be recruited more during feeding on tougher food types. Finally, the capacity to generate force varies greatly over the entire gape cycle, with rabbits working at the lower end of the gape-force relationship. Our data suggest that the masticatory system modulates power output through two mechanisms, primarily through changes in masticatory frequency, specifically modulating the power stroke phase of the chew cycle, while also modulating muscle recruitment. Finally, our preliminary in-situ data suggests the rabbit functions outside the optimal range of the force-gape relationship. 

The dynamic role of rabbit masticatory apparatus during feeding

Bipeds provide a unique study system to address hindlimb allometry both across and within archosaurs and mammals — having size ranges spanning five orders of magnitude yet overlapping near 1 kg body mass for most major clades. Previous work on non-avian and avian theropods shows that hindlimb proportions cluster within 1/9th of available morphospace, centered around a 60:40 tibia:femur isoline. Here we expand on previous work by including bipedal hopping mammals and examining potential drivers of divergence. We investigate 1) within group scaling, 2) phylogenetic signal, and 3) differences between hopping and striding bipeds. Our avian analysis includes 374 species, spanning 74 families and 17 orders. Our mammalian analysis is restricted to habitual bipedal hoppers and includes 47 species distributed across 5 families and 2 orders. Prior to phylogenetic correction, significant hindlimb positive allometry is seen in Anseriformes, Gruiformes, and Palaeognathae, while significant allometry of some individual segments is found in nearly all avian clades. For mammals, significant positive allometry is seen in Macropodiformes but not Rodentia. After phylogenetic correction using PGLS, significant positive allometry persists in the metatarsal segment of Galliformes, Gruiformes, Palaeognathae, and Diprotodontia. (Macropodiformes exhibited positive allometry of the hindlimb, tibia, and metatarsals after PGLS.) Examining segment proportions revealed negative allometry of the femur with respect to total hindlimb length in these same groups. Hence, whether striding or hopping — avian or mammalian, significant positive allometry of the distal segment is a hallmark of both birds and mammals.

Variable scaling of limb segment proportions in bipeds: Does allometry drive divergence?

During flight, insects can make subtle changes to their wing kinematics that in turn have large effects on their aerodynamic forces. To understand and model the above, therefore requires measurements of natural flight with appropriate spatiotemporal resolution and duration. These factors are determined by wingbeat frequency, size, and behaviour, which are limited by trade-offs within the operating equipment. Here, we describe a new and flexible arena setup that uses ten high-speed cameras to record insects flying freely in an optically clear chamber that can scale from the smallest to some of the largest insects. Pulsed far-red LEDs are focused and aligned with the high-speed cameras to provide bright, uniform backgrounds that eliminate motion blur and are beyond the insects’ visible spectrum. Separate, stereo cameras function as triggers, using real-time image analysis when subjects enter the recording volume. These triggers can control additional stimuli, such as a white light, to induce flight manoeuvres at specific times. The resultant footage is coupled with a comprehensive voxel-carving software package to automatically calculate wing and body kinematics. This includes wing deformations in the form of wing torsion, which is important for accurate aerodynamic modelling. To date, we have documented  free-flight in several species, including Calliphora vicina and Drosophila melanogaster at 6,400 fps, and Anopheles gambiae at 12,000 fps. This setup will generate a large database of high-precision insect free-flight kinematics to better our understanding of this diverse class and be used to feed into studies of insect flight control and aerodynamics.

High-precision insect wing and body kinematics acquisition using a new and flexible free-flight arena

Boxfish (Ostraciidae) have peculiar body shapes, with conspicuous keels formed by their bony carapaces. Previous studies have proposed various hydrodynamic roles for these keels, including reducing drag during swimming, contributing to passive stabilisation of the swimming course, or providing resistance against roll rotations. We tested these hypotheses using computational fluid dynamics simulations of five species of Ostraciidae with a range of carapace shapes. The hydrodynamic performance of the original carapace surface models, obtained from laser scanning of museum specimens, was compared with models where the keels had been digitally reduced. The original carapaces showed no reduced drag or increased passive stability against pitch and yaw compared to the reduced-keel carapaces. However, consistently for all studied species, a strong increase in roll drag and roll added mass was observed for the original carapaces compared to the reduced-keel carapaces, despite the relatively small differences in keel height. In particular, the damping of roll movement by resistive drag torques increased considerably by the presence of keels. Our results suggest that the shape of the boxfish carapace is important in enabling the observed roll-free forward swimming of boxfish and may facilitate the control of manoeuvres.

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The alternative gene mtaltnd4: uncovering the mitochondria’s hidden functions

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