Czechia

Gastropods use adhesive locomotion to successfully traverse a range of substrates by adapting the properties of a thin mucus layer. Sample collection methods can disrupt the native mucus structure, so to investigate this system effectively a method for visualising the native deposition of gastropod mucus trails in situ was developed. Optical profilometry and video analysis were utilised to uncover how animal behaviour impacts mucus structure and analysis of the dried trail topography provided insight into mucus alignment and deposition rate.

Our results revealed that dried gastropod trails have a non-uniform deposition profile with ridges visible along both edges of the trail. This phenomenon was independent of specimen size for the slug species Arion vulgaris, Arion flagellus and Limax maximus, but direction and velocity did impact the prominence of the trail ridges. The dried trail topography was the inverse of the convex deposition profile observed in the rehydrated mucus trails of a marine gastropod, indicating a potential difference in mucin deposition or alignment that is independent of the mucus hydration state. When compared to the model slug species, Ariolimax columbianus, the species from this study had thinner dried mucus trails, highlighting potential variations in the cost of adhesive locomotion between species. Our results suggest that mucus deposition rate and animal behaviour have a greater impact on mucus trail topography than species or specimen mass. Moreover, analysis of the dried mucus trails can provide invaluable insight into the relationship between the hydration and mucin costs associated with mucus excretion and adhesive locomotion.

SEB Conference Prague 2024

Trail Watch: Investigating Gastropod Mucus Trails using Optical Profilometry

trails

optical profilometry

mucus

gastropods

technical paper

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Parasites can affect host behaviour and many physiological traits. Changes to host aerobic metabolism are likely responsible for these parasite induced performance alterations. Whole organism metabolic rate is underpinned by cellular energy metabolism driv en most prominently by the mitochondria. However, few studies have explored how mitochondrial enzymatic activities are linked to parasite infection despite being a putative site for metabolic disruptions. Our previous work suggest ed a link between parasite intensity and mitochondrial enzymatic activities in a variety of metabolic pathways including the tricarboxylic acid cycle, oxidative phosphorylation, and lipid oxidation in key organs of wild caught pumpkinseed sunfish ( Lepomis gibbosus infected with trematode and cestode worms . Here, we expand on these results and assess the link between enzymatic activity and parasite infection across multiple populations of L. gibbosus which differ in the prevalence of these parasite s . We measured enzyme activities in several metabolic pathways (TCA cycle, OXPHOS, lipid oxidation and anaerobic glycolysis) in key organs. We found enzymatic activities differed across lakes. Compared to an uninfected l ake, fish from lakes with high parasite prevalence had higher citrate synthase (CS) and carnitine palmitoy ltransferase (CPT) activity in their hearts higher CPT and cytochrome c oxidase (CCO) activity in the ir gills and higher activities of all tested enzymes in the spleen . For the brain, fish from high prevalence lakes had a higher CCO activity but a lower ETS activity. These results suggest that parasite exposure is related to altered energy metabolism at a cellular level mirroring results observed at the whole organism level

Looking at a smaller scale: exploring the link between endoparasite infections and cellular metabolism

Sharks have an unusual physiology, which remains largely a mystery. Opposed to most bony fish, elasmobranchs are isosmotic with seawater, and do so by accumulating extremely high levels of urea as an osmolyte. As urea is chaotropic, elasmobranchs release trimethylamine N-oxide (TMAO) into bodily fluids to protect proteins from urea toxicity, as well as improving resistance to high temperatures and pressures. In addition, elasmobranchs cannot directly oxidise lipids, which are converted to ketones, that are exported to other tissues. Elevated ketones acidify the blood in mammals, and the formation of acetone results in wasted energy. Moreover, in mammals this process is accessed in hypoglycaemic states due to glucose poor diets, starvation, and severe diabetes. To date there has been no work that interdigitates the role and influence of TMAO with energy metabolism in sharks. We also know little about how ketones and TMAO contribute to stressors such as hypoxia. Using high-resolution respirometry and a novel ATP-binding fluorophore, we addressed the effect of TMAO on mitochondrial ketone, lipid, pyrimidine and carbohydrate driven respiration, and ATP production, in cerebellum and heart of anoxia-tolerant epaulette sharks exposed to normoxia and 24h hypoxia.

TMAO regulates mitochondrial metabolism in brain and heart of the anoxia-tolerant epaulette shark Hemiscyllium ocellatum

Natural populations of guppies in the Northern Range Mountains of Trinidad range from diverse, predator-rich communities in larger, higher order streams to small headwater tributaries with only one other fish species and reduced fish of predation. 1970’s vintage life history theory accurately predicts how life histories evolve in these tributaries, where guppies experience reduced mortality risk. They delay maturity and reduce investment in reproduction. The expected tradeoff is that reduced reproductive investment will be accompanied by the evolution of the ability to reproduce to more advanced ages and have extended lifespan. In fact, I found that the opposite was true. Guppies adapted to high predation communities begin to reproduce at an earlier age, invest more in reproduction, continue to reproduce to more advanced ages and have delayed accelerations in mortality risk relative to those adapted to low predation communities. These differences define those from high predation communities as super guppies and leads to the expectation that the low predation life history should never evolve, yet it does. How and why? The answer lies in the indirect consequences of life with and without predators. Predators create a guppy environment where guppy population densities are low and food is abundant. In the absence of predators, guppy populations soar and they deplete the environment of resources. The tradeoffs that favor the evolution of the low predation life history only appear as genotype by environment interactions that include the effects of population density and interspecific competition.

Guppy Life History Evolution - Insights on Costs and Tradeoffs Derived from 40 years of Research

Phenotypic development is strongly influenced by inherited and environmental factors in adaptive and in non-adaptive ways. The outcome and duration of the effects of environmental circumstances, such as exposure to stressful conditions, vary with the severity of the stressor and with the life stage at which they are experienced. Physiological processes, such as stress responsiveness and growth rate can be affected, as can life history traits such as reproductive scheduling and longevity. Inherited factors can also have diverse effects that can span more than one generation. Early life is a particularly sensitive period since the phenotypic architecture is forming at this stage. Parental care can buffer the developing organism from environmental variation, but also give parents the potential to shape offspring phenotype to gain the best fitness outcome. The capacity of parents to buffer offspring from adverse conditions during development can vary with parental age, which can also affect inherited components. I will discuss these effects and present data from experimental work that we have conducted using zebra finches, and from some related work in the wild. 



Intergenerational and transgenerational parental effects: parent and offspring perspectives

Physiologically acclimation to temperature change is hypothesized to evolve in environments with high levels of temperature variation. However, this hypothesis has low explanatory power, meaning other factors must be in play. Behavioral thermoregulation can reduce the body temperature variation that organisms experience and selection on thermal physiology (the “Bogert Effect”). However, the interplay between behavioral thermoregulation and the evolution of thermal acclimation is rarely discussed. I will describe how the Bogert Effect applies to thermal acclimation, and use lizards as models to provide examples of how thermoregulation reduces seasonality in body temperatures and performance relative to null expectations from environmental data.

Behavioral thermoregulation and the evolution of thermal acclimation

Plants, like all living organisms are subjected to mechanical forces arising either from their environment or from within their own body. To sense these forces, plants like animals, have developed mechanosensitive (MS) channels.
MS channels identified and partially characterized in plants belong to the MSL (Mechanosensitive channel of Small conductance Like), MCA (Mid1-Complementing Activity), Piezo, OSCA (hyperosmolality-gated calcium-permeable) and TPK (Two-Pore K+) families. AtMSL10 from the model plant Arabidopsis, homologous to E.coli EcMscS, was the first plant channel to be molecularly identified and shown to be mechanoactivated. It is predominantly expressed in aerial organs but also in root tips. AtMSL10 catalyzes large fluxes of anions. Amongst multiple mechanical constraints generated by their environment, plants must cope with recurring mechanical stimulation produced by wind. We show that MSL10 activity is amplified by oscillatory stimulation at frequencies corresponding to wind-driven oscillations. Therefore, AtMSL10 is proposed to represent a molecular component allowing the perception of oscillatory mechanical stimulations by plants. Recently, we have also characterized a rapidly activated calcium MS channel (RMA) in Arabidopsis. We are currently investigating the role of this channel in root mechanosensing.
Finally, we will stress the need to reintegrate plant MS channel in the cellular-context. Indeed, MS channels described with the patch-clamp technique are characterized in the absence of an extracellular matrix. There is a need to map the membrane tension at the cellular scale in order to specifying where, and under which conditions, plant MS channels operate.


Mechanotransduction through force-gated ion channels in plants

Acute monocytic leukaemia (AML-M5) is a bone marrow disease that affects young children. Astonishingly, the discovery of disease-specific induced pluripotent stem cells (iPSCs) capable of recapitulating human pathogenesis allows us to model this disease in vitro. We had previously generated AML-M5-specific-iPSC (AML-M5-iPSCs) from THP-1 cells obtained from a patient[1]. These AML-M5-iPSCs were then induced with growth supplements to enter haematopoietic differentiation to generate monocytic cells. We noticed that reprogramming transgenes Oct3/4, Sox2 and c-Myc were unintentionally integrated into the genome of AML-M5-iPSC during reprogramming. Out of scientific interest, the effects of transgene integration on drug responses were investigated. Firstly, the monocytic cells were characterised to determine their morphological and phagocytotic properties, and compared with their parental cell line THP-1 across 5-, 10-, 15-, 20- and 25-day post-differentiation. Subsequently, cytotoxic effect of Doxorubicin at 0.5, 1, 2 and 4μM on both cells were investigated with CCK-SK viability assay, and apoptotic effect investigated with cell cycle analysis. 
Our results revealed that in terms of size and morphology, both THP-1 and differentiated monocytic cells remained comparable up to 25 days post-differentiation. Within the same duration, their phagocytotic rates also exhibited no significant differences (p>0.05) when investigated with carboxylate-modified red fluorescent latex beads. However, after 24h Doxorubicin treatment, the IC50 value determined for THP-1 cells was 0.59µM, but the IC50 value for differentiated monocytic cells could not be determined; suggesting that they were significantly (p<0.05) less responsive to Doxorubicin. Upon similar treatment conditions, 92.5±3.9% of THP-1 cells entered late apoptosis stage. However, for differentiated monocytic cells, only 0.3±0.2% entered late apoptosis stage, 37.2±1.5% entered necrosis stage and 62.5±1.6% remained in viable stage. Our results showed that transgenes integration was causing monocytic cells to be resistant to Doxorubicin despite the fact the they should only affect haematopoietic differentiation. In addition, the apoptotic effect of Doxorubicin had also been switched to necrotic effect. More interesting, transgene integration did not seem to alter the morphology and phagocytosis of monocytic cells. We postulate that transgenes Oct3/4, Sox2 and c-Myc interfered with the intercalation of Doxorubicin into the DNA, subsequently altering downstream pathways thereby disrupting cell death via apoptosis. In conclusion, further investigations are warranted to determine the mechanism of reprogramming transgene-induced drug resistance in monocytic cells derived from AML-M5-iPSC.


Integration of reprogramming transgenes into AML-M5-iPSC caused differentiated monocytic cells to be resistant to Doxorubicin

Cold stress is one of the major abiotic stress factors affecting rice growth and development, leading to significant yield loss in the context of global climate change. Exploring natural variants that confer cold resistance and the underlying molecular mechanism responsible for this is the major strategy to breed cold tolerant rice varieties. Here, we show that the natural variations of a SIMILAR to RCD ONE (SRO) gene, OsSRO1c, confer cold tolerance in rice at both seedling and booting stages. OsSRO1c possesses intrinsic liquid-liquid phase separation ability in vivo and in vitro and recruits an AP2/ERF transcription factor and cold stress positive regulator, OsDREB2B, into its biomolecular condensates in the nucleus, resulting in elevated transcriptional activity of OsDREB2B. The OsSRO1c-OsDREB2B complex directly senses low temperature through dynamic phase transitions and regulates key cold response genes, including COLD1. Furthermore, introgression of an elite haplotype of OsSRO1c into a cold susceptible indica rice significantly increases its cold resistance ability. Thus, our work reveals a novel cold stress sensing module and provides a promising gene resource for breeding cold tolerant rice varieties.

The OsSRO1c-OsDREB2B complex senses low temperature to confer cold tolerance via direct regulation of COLD1 in rice

In nature, plants and microorganisms communicate with each other by exchanging different signaling compounds including volatile compounds (VCs), some of which have biostimulating properties. In Arabidopsis, VCs from the fungal phytopathogen Penicillium aurantiogriseum promote growth, photosynthesis and root hair (RH) proliferation and hyper-elongation through mechanisms involving ethylene, auxin and photosynthesis signaling. A striking alteration in the proteome of roots of fungal VC-treated plants involves strong up-regulation of RALF22. To test the possible involvement of the RALF22 in the fungal VC-promoted RH changes, we characterized the RH responses of ralf22 and fer-4 plants impaired in RALF22 and its receptor FERONIA to VCs emitted by P. aurantiogriseum. We found that these plants were unresponsive in terms of VC-promoted RH elongation and proliferation. Unlike in WT roots, fungal VCs did not enhance the transcript levels of RALF22 in roots of fer-4 and ethylene- and auxin- insensitive mutants, and those of RH-related, ethylene-responsive genes (e.g. RSL4, RHD2, PRX1 and PRX44) in ralf22 and fer-4 roots. Finally, we identified ethylene as the bioactive fungal VC, as VCs of ΔefeA strains of P. aurantiogriseum cultures impaired in ethylene synthesis weakly promoted RH proliferation and elongation in exposed plants. Collectively, our results demonstrate that RALF22-FERONIA complex is a key determinant of the ethylene, auxin and photosynthesis signaling-mediated RH response to fungal ethylene emissions.

RAPID ALKALINIZATION FACTOR 22 is a key modulator of the root hair growth responses to fungal ethylene emissions in Arabidopsis

Human cultivation during plant domestication imposed strong selection
on fitness relevant traits. The relationship between apparent trait
changes and fitness is not always obvious. For instance, seed color
changed in numerous crops early during their domestication. In the
pseudocereal grain amaranth seed color changed from dark to white
three times during its repeated domestication. However, the genetic
basis and functional relevance of the seed color change is still
unknown.
To study the ecological function of seed pigments in grain amaranth,
we assessed germination properties in hundreds of lines using a
semi-automated phenotyping system and found that white seeds germinate
significantly faster than dark seeds. The metabolomic analysis of
seeds showed reduced accumulation of flavonoids, including the dark
proanthocyanidins, in white seeds. To study the genetic control of the
change, we employed genome-wide association mapping and RNA sequencing
in diverse accessions. Differential gene expression analysis revealed
the downregulation of almost all flavonoid pathway genes in white
seeds. The genomic analyses identified the insertion of a transposable
element (TE) into a regulator of the flavonoid pigment pathway as one
causal mutation for the seed color change. Using full-length
transcript sequencing, we show how the TE induces alternative splicing
in the regulator which results in loss of function for pigment pathway
regulation. We demonstrate how a seemingly fitness-unrelated trait
like seed color can confer environmental adaptation and how the
insertion of a transposable element altered the expression of a whole
metabolic pathway to enable the trait change.

Trail Watch: Investigating Gastropod Mucus Trails using Optical Profilometry

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Trail Watch: Investigating Gastropod Mucus Trails using Optical Profilometry

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Looking at a smaller scale: exploring the link between endoparasite infections and cellular metabolism

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