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Antarctic fishes are extreme stenotherm animals, adapted to live at 
temperatures close to the freezing point of ocean water (−1.9 °C). 
They experience stable annual temperature fluctuations less than 1 
°C. Due to climate changes and the consequent increment in water 
temperature, Antarctic species are exposed to stressful conditions. The 
effects of increasing ocean water temperature on morpho-functional 
traits of Antarctic marine species is under attention. In this study we 
evaluated the effects of acute heat stress on the skeletal muscle of 
both the Antarctic haemoglobinless Chionodraco hamatus and the 
red blooded Trematomus bernacchii at morphological and molecular 
level (cell death, heat shock response, antioxidant defense and NOS/ 
NO system). Our preliminary results suggest a specie-specific morphofunctional 
response of the skeletal muscle of the two Antarctic teleosts 
to heat stress.

SEB Centenary Conference 2023 

Effects of heat stress on the skeletal muscle of Antarctic Trematomus bernacchii and Chionodraco hamatus

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The African dipnoi Protopterus dolloi is an obligate air-breathing fish re-thickening of epidermal layers. Immunofluorescence reveals that
aestivation correlates with an increased oxidative stress and changes
of Heat Shock Proteins expression, suggesting a protective role for
these chaperons. Our findings revealed that lungfish skin undergoes
remarkable morphological and molecular readjustments in response
to stressful conditions associated with aestivation.
that, during dry season, experience a period of dormancy named
aestivation. Aestivation is characterized by complete reliance on
pulmonary breathing, decrease of metabolism and down-regulation
of respiratory and cardiovascular functions. To date, little is known
about morpho-functional rearrangements induced by aestivation
in the skin of African lungfishes. Our study aims to identify, in the
skin of P. dolloi, structural changes and stress-induced molecules in
response to short-term (6 days) and long-term (40 days) aestivation.
Light microscopy showed that short-term aestivation induces a
narrowing of epidermal layers and a decrease of mucous cells while
prolonged aestivation is characterized by regenerative processes and

The skin of lungfish Protopterus dolloi under freshwater and aestivating conditions

Fishes exhibit an extraordinary array of locomotor behaviours, from 
swimming with their pectoral fins, to body undulations, to leaping 
outside the water and walking on the substratum. Different fishes can 
both walk and swim underwater. Fishes that evolved walking might 
benefit from energy saving at low speeds by avoiding lift-induced drag 
and allowing adherence to the substratum in turbulent conditions. 
These fishes may display varying preferences for walking and swimming 
which correlate to the shape and size of propulsors and locomotor 
behaviour. In fact, walking evolved in highly different fish taxa and 
involves an unexplored diversity of anatomy and morphology of fins, 
and movements. In this project, we aim at quantifying the diversity of 
walking behaviour. We investigate the kinematics of swimming and 
walking in a range of bony and cartilaginous fishes to uncover a general 
pattern of how and why some fishes exhibit a walking behaviour. Fishes 
are recorded during walking and swimming across different speeds in a 
flow tank using two synchronized, orthogonal high-speed cameras. For 
each species, we extract video-based parameters of body undulations 
and pectoral fin movements. We further compare different metrics 
retrieved from movement parameters across the species of interest 
to uncover general patterns of how and why fishes walk underwater.

How and why fishes walk: a biomechanics perspective

Syngnathids (seahorses, pipefishes, and seadragons), are rare in 
that embryos develop on or in the male in a specialised brooding 
structure. Seahorse brood pouches provide post-fertilisation nutrients 
(patrotrophy) in the form of lipid transport during pregnancy. We 
tested the hypothesis that protein, vital for gene regulation and tissue 
growth during embryogenesis, is also transported to developing 
embryos during pregnancy in the Australian pot-bellied seahorse, 
Hippocampus abdominalis. We used dry masses and total nitrogen 
content to estimate total protein contents in recently fertilised embryo 
and newborn H. abdominalis. There was a significant increase in 
protein mass from recently fertilised embryos to neonates. The 
significant increase in protein mass over embryogenesis suggests that 
paternal protein transport occurs during H. abdominalis pregnancy. As 
protein yolk reserves are depleted by embryonic metabolism, paternal 
protein supplementation may be required. This study deepens our 
knowledge of the intimate relationship between developing embryos 
and the male placenta and furthers understanding of the paternal 
nutritional contribution to embryonic development in male pregnant 
vertebrates. Keywords:brood pouch, embryonic provisioning, nitrogen, 
parentotrophy,paternal investment, reproductionReferences [1] 
Z. M. G. Skalkos, J. U. Van Dyke, C. M. Whittington, J. Comp. Physiol. B: 
Biochem. Syst. Environ. Physiol.,190547-556, (2020). [2] J. U. Van 
Dyke, O. W. Griffith, M. B. Thompson, Am. Nat.,184, 198-210, (2014).

Evidence of paternal protein provisioning during male pregnancy in the seahorse, Hippocampus abdominalis

In vitro experiments indicate that Cymbopogon citratus possesses 
antioxidant and anti-dyslipidemia activity. This study tests the 
hypothesis that Cymbopogon citratus prevents fructose-induced 
dyslipidemia in adult rats. Male Sprague-Dawley rats were fed normal 
rat chow and drank water alone (C), 10% fructose in water (F), 20% 
Cymbopogon citratus extract in water (CC) or 10% fructose plus 20% 
Cymbopogon citratus extract in water (FC) for 8 weeks. At 14 weeks of 
age, rats were collected blood samples. All data are expressed means ± 
S.E.M., compared by ANOVA, body weight, blood urea nitrogen (BUN), 
plasma creatinine (Cr), plasma aspartate aminotransferase (AST), 
plasma Alanine transaminase (ALT), plasma triglyceride and plasma 
high-density lipoprotein were not significantly different among the four 
groups. Compared to C group, high fructose intake (F group) significantly 
increased fasting blood, oral glucose tolerance test, plasma cholesterol and plasma low-density lipoprotein. These fructose effects were all 
abolished by Cymbopogon citratus extract treatment (FC group). In 
addition, the Cymbopogon citratus extract treatment alone had no 
any significant effect on measured parameters. The present data 
indicate that Cymbopogon citratus extract prevents fructose-induced 
dyslipidemia in adult rats, likely by increasing lipid metabolism.

Cymbopogon citratus prevents fructose-induced dyslipidemia in rats

Age-related increase in oxidative stress plays a key role in cognitive 
decline. Regular exercise and an essential amino acid Taurine 
supplementation are effective against the progression of oxidative 
stress in age-related degenerative changes. Thus, this study 
investigated the effects of taurine with exercise on antioxidant enzyme 
activities in middle-aged rat brains. Male rats (15 months old) were 
divided into sedentary and swimming exercise groups. Rats received 
water, 1% tween-80, vitamin E (50 IU/kg), or taurine (800 mg/kg) for 
8 weeks. Antioxidant enzymes (SOD, CAT, and GPx) activities in the 
basal forebrain, cerebral cortex and hippocampus were determined. 
Exercise significantly increased GPx activity levels in the basal forebrain, 
compared to the sedentary group. In the sedentary group, taurine 
significantly increased SOD levels in basal forebrain and GPx activity 
levels in the hippocampus, compared to the control group. In the 
exercise group, taurine significantly increased SOD levels in the cerebral 
cortex, CAT and GPX activity levels in the hippocampus, and CAT and 
GPX activity levels in the basal forebrain, compared to the control 
group. Taurine supplement in conjunction with exercise significantly 
increased CAT and GPX activity levels in the hippocampus, compared to 
taurine supplementation in the sedentary group. Taurine supplement 
in conjunction with exercise is capable of reduction of oxidative stress 
and enhancement of antioxidant enzymes activities in middle-aged 
rat brains. Therefore, taurine supplements in conjunction with exercise 
may benefit in the reduction of oxidative stress which is contributed 
to age-related neurodegenerative diseases.

Effects of taurine supplementations with exercise on antioxidant enzyme activitiesin middle-aged rat brains

Stress and elevated glucocorticoids, particularly cortisol in teleost
fish, have been linked with pathological remodeling of the heart, and deterioration of fish fitness and welfare. However, these associations
were based on biomarkers that fail to provide a retrospective view
of stress. This study is the first to unravel the association of cortisol
in scales, as chronic stress biomarker, with cardiac morphology
and whole-animal performance of Atlantic salmon (Salmo salar).
Growth, heart morphology, plasma and ontogenetic scale cortisol
levels, and expression of genes involved in cortisol regulation of
the hypothalamic-pituitary-interrenal (HPI) axis of undisturbed fish
(CONTROL) were compared to fish exposed daily to random stressors
(STRESS) for 8 weeks. Though scale cortisol levels showed a timedependent
accumulation in both treatments, the cortisol levels of
STRESS fish in plasma and scales were 29.1% and 25.0% lower than
CONTROL, respectively. These results can be attributed to the significant
upregulation of both glucocorticoid and mineralocorticoid genes (gr
and mr) involved in the negative feedback in STRESS treatment, coupled
with the lack of strong indication for excess de-novocortisol synthesis
manifested by the stable crh, pomcs , and star gene expression. The
cortisol levels are, however, counterintuitive to the growth performance
as STRESS fish grew 33.7% slower than CONTROL. Moreover, STRESS
fish had 8.4% relative ventricle mass (RVM), a maladaptive response
documented in cortisol-exposed fish. Nevertheless, heart histology
showed comparable compact/spongy myocardium proportion and
extra-bundular sinus area between the treatments, which suggests
heart growth as an adaptive response to enable fish to survive (chronic)
stressful conditions.

Associating chronic stress, quantified by scale cortisol, with growth reduction and heart remodeling of Atlantic salmon (Salmosalar)

Calibration for quantifying the strontium-to-calcium ratios of aragonitic shells using EPMA with WDS

Biogenic calcium carbonate structures such as molluscan shells and 
fish otoliths contain >30 of minor and trace elements. The elemental 
composition is often quantified by using Electron Probe Micro Analyzer 
(EPMA) with the wavelength dispersive X-ray spectrometer (WDS), 
Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LAICP- 
MS), and/or Secondary Ion Mass Spectroscopy (SIMS). EPMA 
provides reliable data even by emitting the electron beam with a 
diameter of 1 μm, and may be superior to LA-ICP-MS in terms of 
spatial resolution. However, X-ray intensity output from EPMA is quite 
dispersive, which means that one has to average out the measurement 
error by repeatedly observing the same position of a sample surface to 
improve the precision. Here, we investigated the statistical properties 
of the output from EPMA with WDS, by analysing Monetaria annulus 
shells prepared by rearing wild-collected individuals at constant 
temperatures. We investigated 1) the effect of probe current, beam 
diameter and/or beam exposure time on measurement precision and 
2) the effect of repeatedly focusing the electron beam on the same 
spot of a sample surface on X-ray intensity.

Statistical properties of X-ray intensity output from EPMA with WDS for quantifying the strontium concentration in aragonitic shells

Environmental stress like injury or the exposure to toxic metals like 
Cadmium (Cd) are energy demanding processes. A previous study 
suggested that Cd detoxification is inhibited in injured earthworms, 
which could be due to limited energy sources. Hence, we examined in 
the present study different energy parameters in tissue of Lumbricus 
terrestris exposed to Cd with and without injury over a period of 
three weeks. The most prominent differences revealed the group of 
earthworms that were injured and at the same time exposed to Cd. We 
revealed significant changes in amp-activated protein kinase (pAMPK) 
levels, the main energy sensor of the cell as well as 70 kilodalton 
heat shock protein (HSP70), a molecular chaperone known to be 
involved in the innate immune response. Additionally, we measured 
glucose and protein levels, which changed significantly after one 
and two weeks, while after three weeks no significant differences 
could be observed between the treatment groups. We conclude that 
injured earthworms exposed to Cd have a higher energy demand than 
earthworms exposed to a single stressor. When we looked at injury 
and Cd exposure separately, we observed that these stressors caused 
different changes in pAMPK and HSP70 levels especially during the 
first week of treatment revealing stress-specific effects, which need to 
be further explored in future studies. However, we could not confirm 
the hypothesis that detoxification processes are inhibited in injured 
earthworms due to limitations in energy provision.

Effects of injury and cadmium on the energy status in the earthworm Lumbricus terrestris

Climate change has increased the frequency of extreme weather and led 
to abnormal climate annual variation, especially having a major impact 
on aquaculture in East and Southeast Asia. The frequent temperature 
spikes in summer, which exceed the optimal range of the organisms, 
immune system disorders of organisms in tropical areas. Resveratrol 
(RSV) is a polyphenols compound that alters the pro-/anti-inflammatory 
activity against foodborne pathogens. In this study, we hypothesize 
that the RSV-supplemented diet can against bacterial infection and 
prevents the inflammation of aquaculture animals in summer. At first, 
the toxicity of RSV was tested in zebrafish embryos, and it revealed that 
RSV had no detrimental effects and did not affect their survival rate. 
And therefore, tropical tilapia (Oreochromis mossambicus) were used 
as test subjects to determine the effect of an RSV-supplemented diet 
on the fish gut microbial composition. The result of PacBio 16s rRNA 
sequencing indicates that RSV can reduce the potential pathogenic 
bacteria (AeromonasandPlesiomonas) and increase the abundance of 
probiotics (Cetobacterium) in the gut and gills of tilapia. In addition, 
using RSV to perform the minimum inhibitory concentration (MIC) test, 
we found RSV can inhibit pathogenic bacteriain vitro. Furthermore, we 
will investigate the effect of microbial on various physiological and 
immune responses of tropical fish after feeding RSV by the RNA-Seq 
analysis. Accordingly, the results indicated that RSV has the potential to 
serve as a valuable dietary supplement for tropical fish to cope with the 
frequent temperature spikes in summer, as a result of climate change.

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Effects of heat stress on the skeletal muscle of Antarctic Trematomus bernacchii and Chionodraco hamatus

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The skin of lungfish Protopterus dolloi under freshwater and aestivating conditions

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