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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.  

SEB Conference 2022

De novo assembly of the crucian carp genome

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.

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.
 



GM plants 2.0: Can DNA nanotechnology enhance plant genome engineering?

The structural integrity of biological armours derives in part from their geometric architectures. Armours often involve tessellations of self-similar structural elements, tiled together to form a shell-like surface. The boxfish carapace, for example, is comprised of polygonal mineralized dermal plates, called scutes, which are arranged in a complex geometric pattern. Unlike artificial armours, the exoskeleton grows along with the fish. Therefore, the relationship between the tessellation and the overall structure of the armour is critical to maintain protection throughout growth. Using high-resolution microCT data and segmentation algorithms, we quantify architectural aspects for each of the hundreds of scutes that build the armour, across an ontogenetic series of the longhorn cowfish Lactoria cornuta (from 1.7 cm to 13.6 cm body length), to investigate the maintenance of the tessellated structure throughout growth. We show that instead of adding scutes to enlarge the carapace surface, scute number remains constant, and carapace growth is achieved by scutes increasing in absolute volume, thickness, and especially width. Scutes also become relatively thinner and in areas of more complex topology (e.g. around fin insertions, mouth, anus) smaller scutes with more variable curvature are found. Besides providing insights into developmental constraints of this species related to carapace growth, our findings might also provide perspectives into natural strategies for construction of mutable tiled architectures.

Carapace growth of the longhorn cowfish Lactoria cornuta: A tessellated armour through ontogeny

Metabolic rate is a fundamental trait that influences and constraints the behaviour and performance of animals. However, conventional measures of metabolic rate, based on whole-animal oxygen consumption, often fail to show the predicted relationships with measures of animal performance since it is unclear what proportion of consumed oxygen is associated with ATP production. Thus the efficiency with which the mitochondria convert oxygen into ATP can be a better determinant of an animal performance capacity. In this study we examined whether mitochondrial efficiency predicts sustained swimming performance in the European minnow (Phoxinus phoxinus), a riverine fish. We measured individual critical swimming speed (Ucrit) followed by a measurement of maximum metabolic rate (MMR), standard metabolic rate (SMR) and mitochondrial function, predicting that measures of mitochondrial performance such as the ATP produced per molecule O2 consumed will be positively related to Ucrit and that the variation in Ucrit explained by mitochondrial function will be greater than any explained by MMR and SMR. Maximal rates of oxidative phosphorylation at the mitochondrial level were positively correlated with maximal oxidative metabolism of the whole fish. However, contrary to our predictions, swimming performance was unrelated to both organism-level and our measures of mitochondrial level metabolism, with Ucrit, MMR and SMR being influenced by individual body mass only. This suggests that, while mitochondrial function predicts whole-animal metabolism, it does not necessarily constrain animal locomotor performance, and the maximum sustained swimming speed is not determined by energy or oxygen supply. Further research is needed to determine the limits to physical performance.

Sustained swimming performance is independent of organism-level and mitochondrial-level metabolism in European minnows.

Mammalian cell culture is widely used for discovery and development. Recently, increasing
attention has been paid to the importance of maintaining physiologically-relevant conditions in cell
culture. Although oxygen level is a particularly important consideration, it is rarely regulated by
experimentalists. The atmospheric O2
levels commonly used in cell culture are significantly higher
than those experienced by most mammalian cells in vivo, leaving cells susceptible to oxidative damage, senescence, transformation, and otherwise aberrant physiology. A barrier to incorporating O2
regulation into most cell culture workflows has been the expense of investing in new equipment, as
the vast majority of laboratory CO2
incubators do not regulate O2
. Here, we describe an inexpensive
(<CAD 1000), portable and user-friendly O2/CO2
incubator that can establish and maintain physiological O2
, CO2
, and temperature values within their physiological ranges. We used an Arduino-based
approach to add O2 and CO2 control to a temperature-regulating egg incubator. Our incubator
was tested against a commercial laboratory O2/CO2
incubator. Using Presens OxoDish technology, we demonstrate that at a setpoint value of 5% gas-phase incubator O2
, media O2 averaged
5.03 (SD = 0.03) with a range of 4.98–5.09%. MCF7, LNCaP and C2C12 cell lines cultured in the
incubator displayed normal morphology, proliferation, and viability. Culture for up to one week
produced no contamination. Thus, our incubator provides an inexpensive means of maintaining
physioxia in routine mammalian cell culture.

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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

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