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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.

SEB Conference 2022

Distinguishing de novo protein synthesis from protein turnover with rapamycin to inhibit the mTORC1 pathway in the European perch.

poster

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.

Many flying animals such as birds, bats and insects are able to navigate through cluttered environments and have a robust guidance strategy to avoid crashes and collisions. Some of their most impressive behaviours include flying rapidly through small gaps, dodging obstacles, and perching on moving objects. Lab studies have shown that many diurnal birds and insects use visual guidance strategies relying on optic flow to avoid obstacles when flying;a however, dense natural environments such as forests are visually cluttered which can make integrating information from optic flow a complex cognitive task. Therefore, optic flow could be used in conjunction with other cues, such as contrast between obstacles and their background. This project aims to investigate the possibility that birds use a strategy based on relative brightness to stay clear of edges when negotiating gaps, as was found in a study by Baird and Dacke in orchid bees. 
Zebra finches were presented with different brightness gradients behind the aperture of a tunnel. The gap negotation strategy of the birds as they were flying through the aperture was not directly affected by alternating the contrast; however, the birds' decision of whether to fly through the tunnel, as well as the exit direction taken after flying through it, were both influenced by the different brightness gradients. This hints at a possible pre-planning of the trajectories before the onset of flight, where birds approach obstacles after having already established their flight strategy.

Do birds use contrast cues for visual guidance during flight?

Metabolic scaling describes the relationship between metabolic rate and body size, and can be calculated using the equation Metabolic Rate = a(Body Mass)B.  Juvenile mammals have been reported to have higher exponent (B) values until they reach adult body size experience a critical switch, after which B decreases and scaling is hypometric. This study measured and compared metabolic rate and mass in 13-lined ground squirrels (Ictidomys tridecemlineatus) through a wide range of developmental stages to investigate how mass specific metabolic rate (MSVO2) change throughout parturition (change in metabolism with no change in weight), as well as how MSVO2 scale as pups gain weight though development (weight gain due to growing tissue). Metabolic rates of 6 litters were found to increase roughly 150% upon birth. Developing I. tridecemlineatus pups then displayed a switch from hyper- to hypometric scaling at 20-26 days of age, as marked by a decrease in the exponent B value from 1.20 to 0.76 (adj R2=0.90, p p=2.70E-03; adj. R2=0.67, p=2.84E-02). The critical switch in B was found to occur before reaching adult size, thus indicating factors other than body size may be more influential for scaling during development.

Changes in Metabolic Scaling Throughout Development in Small Mammals

The crucian carp (Carassius carassius) is a champion of anoxia tolerance and the physiological mechanisms are well documented. Still, there is a need for better understanding at the molecular and genetic level, and a fully sequenced, assembled and annotated genome will  facilitate such studies. To obtain a high-quality genome assembly, we sequenced the crucian carp genome using methods such as chromosome conformation capture (HiC) and PacBio long-read sequencing, in addition to short-read sequencing for error correction. For annotation we are using full-length transcripts (IsoSeq data) as well as regular RNAseq data from both normoxic, anoxic and re-oxygenated crucian carp. The genome of crucian carp and other cyprinids consists of 50 chromosomes that originated from several whole-genome duplication events, with the most recent occurring 14 Mya. The presence of duplicated genes offers the possibility for evolutionary changes through the mechanism of expression dominance: one paralog remains most expressed, while the other paralog is free to change without compromising the expression integrity of the biological systems required for normal function. This is hypothesized to be the genetic mechanism having allowed the crucian carp and goldfish (Carassius auratus) pyruvate dehydroxylases to obtain the decarboxylation function necessary for conversion of pyruvate to acetaldehyde; an important metabolic process in the crucian carp’s survival strategy in anoxia. We expect that paralogs of other important genes may have undergone similar changes, and aim to elucidate any differences that may have arisen between the crucian carp and the goldfish due to the long domestication history of the latter.  

De novo assembly of the crucian carp genome

In estuaries, flooding of natural or anthropogenic origin cause a sharp decrease in salinity and pH as well as an increase in turbidity that can be maintained for several days. Although salinity is the main biogeographical determinant in these ecosystems where it changes along a dynamic gradient, the responses of benthic intertidal communities to intense hypoosmotic stress may differ from those caused by daily and seasonal salinity variations that are typically studied. In addition, the response of these communities to several combined stressors encountered during floods (decreased salinity and pH, increased turbidity) has rarely been studied. Here, we aim to evaluate the relative sensitivity of three benthic macroinvertebrate species (Littorina saxatilis, Limecola balthica and Mytilus spp.) to conditions mimicking spring flooding. We measured the survival rate of organisms exposed to a gradient of periodic exposure (12 treatments, characterized by a period of exposure lasting 0 to 9 days interspersed with 24 hours of exposure to marine salt water, over a continuous six-weeks cycle) in acidic and turbid freshwater (pH 5.7, 60 NTU), and in untreated freshwater (pH 7.7, 0 NTU). For the three species, the mortality was higher in the treatments in the acidic and turbid water, and in the treatments with the longest period of exposure. L. saxatilis showed the highest mortality rate, followed by L. balthica. Mytilus spp. showed almost no mortality. Longer and more frequent flooding might have an impact on estuaries invertebrate communities. Our study also highlights the importance of considering combined factors that are more representative of natural conditions.

Potential marine benthic macro-invertebrates’ responses to flooding: in vitro responses to a combination of freshwater exposure, low pH and high turbidity on three intertidal mollusks

Musculoskeletal lever systems are typically regulated by antagonistic muscle pairs. Some of these systems are involved in mechanically varying task. For example, bird beaks exert high static forces while biting and move the beak up-and-down at high velocities during food handling and vocalisation. As increased bite force evolves, the closer muscle force will become multiple times stronger than the opener muscle force. This has a direct effect on high frequency movement performance, as fully activating the closer muscles will impede beak opening while the closer muscles are relaxing. Muscle fibre type composition could influence the frequency further as slower fibres have considerably longer relaxation times. To investigate how muscle activation patterns should be regulated to achieve maximal beak frequency, we constructed a simple forward dynamic model of two antagonistic muscles pairs, both with fast-twitch and slow-twitch muscles, which rotate a lever back and forth in MATLAB Simulink. Morphological model parameters are based on measures from a songbird (Padda oryzivora) and muscle contractile properties were taken from the literature. The model shows that fibre type, especially those from the most forceful (closer) muscle group, has a strong effect. To achieve maximal frequencies, models using only slow muscle fibre types should twitch larger parts of their closer muscle (13.4% vs. 4.6%) and will inevitably achieve lower beak-tip frequencies (13.45 vs. 27.95 Hz) than models with fast muscle fibre types. We will discuss how an orderly recruitment of motor units in muscles consisting of mixtures of fibre types affects the performance.

The effect of muscle fibre type on high-frequency beak movement: a forward dynamic modelling approach

Locomotion is essential for the survival and fitness of animals. Fishes have evolved a variety of mechanisms to minimize cost of transport. For instance, bluegill sunfish have recently been shown to employ intermittent swimming in nature and in laboratory conditions. We focused on understanding the functional properties of the power-producing muscles that generate propulsive forces in bluegill to understand the implications of intermittent activity. We used in vivo aerobic muscle activity parameters (e.g. oscillation frequency, muscle strain, timing of activation) in muscle physiology experiments to examine muscle power output during intermittent vs. steady swimming in these fish. Intermittent propulsion involves swimming at relatively slow speeds with short propulsive bursts alternating with gliding episodes.  The propulsive bursts are at higher oscillation frequencies than would be predicted for a given average swimming speed.  The work-loop muscle physiology experiments demonstrated that intermittent activity allows muscle to produce sufficient power for swimming compared to imposed steady swimming conditions. Further, the intermittent muscle activity in vitro reduces fatigue relative to continuous activity. This work lends support to the fixed-gear hypothesis that suggests that there are preferred oscillation frequencies that optimize efficiency in muscle use and minimize cost of transport.

Muscle Power Production during Intermittent Swimming in Bluegill

Geckos are skilled climbers, and can even scale smooth surfaces, due to powerful adhesive structures on their feet. Gecko adhesive toepads are activated when pulled towards the body, but readily detach when pushed away from it. This direction-dependence allows rapid control of attachment but presents a problem when climbing head-down. 
To study the kinematic and dynamic alterations with direction, we extracted kinematics and 3D-ground-reaction-forces for six individuals of geckos (Hemidactylus frenatus), climbing head-up or head-down on a vertical racetrack. 
Based on previous work, we expect the adhesive role of the feet to swap when climbing head-down. We proposed three hypotheses for mechanisms likely to be employed:
(1) Hind limbs are rotated backwards, aligning the activation direction of the adhesives with the direction of the gravitational force – (2) diagonally opposed foot angles sum up to 180 ° generating inward forces of opposite orientation – and (3) toes are spread further apart with individual digits pulling.
We found that hind feet were rotated further backwards for head-down climbing, increasing the relative adhesive force. While the sum was 173°, the direction of the force vectors did not oppose each other, and adhesive forces did not increase with greater diagonal angles. Finally, against expectations the toe spreading in hind feet decreased for head-down climbing, but not increasing relative adhesive forces significantly.
By setting kinematic adjustments in context with forces produced we determined which kinematics are good predictors for generating adhesion, representing a valuable insight into climbing locomotion and application of bio-inspired gaits to legged robotics.

Multilevel exploration of kinematic and dynamic adjustments of geckos (Hemidactylus frenatus) climbing vertically up and down

Carnivorous Nepenthes pitcher plants normally use motionless pitfall traps with slippery walls to catch their prey. Nepenthes gracilis uniquely exploits rain drop impacts on the roof-like pitcher lid to flick insects into the pitcher. This mechanism relies on three independent adaptations of the lid: a horizontal orientation, an attachment-reducing wax crystal coating of the lower lid surface, and high stiffness of the lid itself combined with an elastic ‘hinge’ that facilitates a torsion spring-like impact response. Previously, it has been postulated that this elastic element is located in the ‘neck’ region between pitcher and lid.  We superimposed three serial micro-computerised tomography (µ-CT) scans with different lid positions to show an asymmetric deformation reaching far into the pitcher body. Downward lid displacement deformed two distinct areas in the neck and the pitcher body, while upward displacement stretched the entire dorsal spine of the pitcher uniformly. Cross-sectional slices and densities from the µ-CT scans  showed that areas of maximal deformation coincide with local minima of the second moment of inertia, while stiffer areas are geometrically and structurally reinforced. Load-displacement trials revealed strongly asymmetric spring properties, with 83% larger downward than upward lid displacement under equal loading. Our results point to a key role of pitcher geometry for effective ‘springboard’ trapping in N. gracilis.

Disentangling the influence of geometry and material properties on the 'springboard' trapping mechanism in carnivorous Nepenthes gracilis pitcher plants

Male mosquitoes detect and localise conspecific females by their flight tones using the Johnston's organs (JO), which detect antennal deflection under the influence of local particle motion. Acoustic behaviours of mosquitoes and their JO physiology have been investigated extensively within the frequency domain, yet the auditory sensory range and the behaviour of males at the initiation of phonotactic flights are not well known. In this study, we predict a maximum spatial sensory envelope for flying Culex quinquefasciatus by integrating the physiological tuning response of the male JO with female aeroacoustic signatures derived from numerical simulations. Our sensory envelope predictions were tested with a behavioural assay of free-flying male mosquitoes responding to a female-like artificial pure tone. The minimum detectable particle velocity observed during flight tests was in good agreement with our theoretical prediction formed by the peak JO sensitivity measured in previous studies. The iso-surface describing the minimal detectable particle velocity represents the quantitative auditory sensory range of approaching males and is directional with respect to the female attitude. Our results illuminate the intricacy of the mating behaviour dynamics and point to the importance of observing the attitude of flying mosquitoes to understand fully the sensory ecology of conspecific communication.

Auditory sensory range of male mosquitoes for the detection of female flight sound

Gene targeting (GT) is a method of precise genome engineering that can facilitate crop improvements that are central to meeting growing global food demands. Yet GT frequencies are very low in higher plants, mainly due to the low frequency of homologous recombination (HR) upon which GT relies, hindering realisation of the technology’s potential. Despite advancements, routine GT frequencies remain ~ &lt;1% and random integration of donor DNA and GT machinery causes problematic off-target effects; therefore, further improvements in GT frequency and accuracy are required. Here, a novel approach to address these problems is described, based on the application of DNA nanotechnology to GT in plants. In this approach random integration of the GT template will be prevented by concealing the free dsDNA ends of the repair template with nanostructure ‘caps’, as we propose that it is the free dsDNA ends that serve as the substrate for random integration. Here I report that capped structures containing a GFP reporter gene have been successfully transformed into plant cells via Agrobacterium and GFP expression detected on confocal microscopy. This demonstrates that these structures get into the plant cell nucleus and do not hinder enzymatic access to the gene, an important milestone in developing this technology. To test the nanostructure’s ability to improve GT efficiencies and reduce off target effects, an assay was established to measure GT rates with or without nanostructures. GT events have been successfully visualised by GFP expression in plant cells using confocal microscopy and flow cytometry. Next steps are to assay the rates of random integration using the nanostructure caps via qPCR-based assays and  to design, build and test the next generation of DNA nanostructures for prevention of random integration using DNA origami nanotechnology as a transgene delivery vehicle. Overall, this project brings together two exciting technologies to address the need for continued improvement in GT frequencies and preventing random integration. This will facilitate the improvement of plant genome engineering tools that can deliver vital improvements to crops required to bring sustainable agriculture into the 21st Century.
 



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Do birds use contrast cues for visual guidance during flight?

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