The phenotypic plasticity of thermal tolerance and its modulation in a complex environmental scenario

It is well established that climate change constitutes a great challenge for wild animals, with increasing occurrence of heat waves also causing mortality events. However, in nature, environmental stressors are not occurring alone, but several occur at the same time. Among common environmental stressors, presence of trace metals is one of the greatest threats for aquatic animals because of their persistence in the environment and the bioavailability for organisms. Organisms can improve their tolerance to stressors via phenotypic plasticity, i.e., the ability of an organism to change phenotype in response to stimuli from the environment, and this ability might also be transferred to the next generations, through parental effect. In the current doctoral thesis, I investigate how fish respond to extreme environmental events via phenotypic plasticity and via parental effects using laboratory and field approaches. In Study I, the focus was to elucidate whether the relationship between transcription and translation of the heat shock proteins was shaped by the magnitude of the heat stress in a laboratory-reared zebrafish population. The study showed a coupling of the transcription and translation at high temperature, and an uncoupling at mild temperatures, highlighting the importance of studying the response of fish to heat stress at protein, and not only at gene level. In the Study II, III and IV, the phenotypic plasticity of thermal tolerance has been individually assessed at both physiological and molecular level, and in two fish species: one temperate species caught from the Baltic Sea (three-spined sticklebacks), and one originally tropical but currently largely laboratory-reared zebrafish. The thermal tolerance of individuals has been measured before and after an acute exposure to heat wave, copper, and combination of the two stressors. In these studies, I observed that the innate thermal tolerance, as well as its phenotypic plasticity, depends on fish species and is shaped by sex, with male stickleback generally showing better plasticity compared to females; but the opposite trend was seen in zebrafish. In sticklebacks, the exposure to heat wave and copper separately, resulted in an increase of the thermal tolerance, therefore suggesting positive phenotypic plasticity. On the other hand, while the heat wave improved the thermal tolerance of control zebrafish, the same was not observed in the copper exposed group. The response observed in Study I at molecular level, was not replicated in sticklebacks’ studies, except for Study IV, suggesting that the molecular response might be also species-specific. In Study IV, the stressor effects were also assessed in the offspring generation. Despite some limitations, Study IV showed that the offspring was strongly affected, as the heat wave and copper negatively influenced the development and thermal tolerance via both developmental and parental exposure. This research represents an important step forward in the study of the thermal tolerance as it addresses its individual phenotypic plasticity and potential transgenerational occurrence in response to multiple-stressor exposure. The approach used in the current thesis was multilevel, spanning from the biochemical response to the evolutionary potential for adaptation, therefore representing a broad focus on the phenotypic plasticity of thermal tolerance.Lämpötilatoleranssin fenotyyppinen plastisuus ja sen vaihtelu kompleksissa ympäristöolosuhteiden muutoksissa Ilmastonmuutos on huomattava uhka villeille eläimille ja lisääntyvät lämpöaallot saavat aikaan jopa eläinten massakuolemia. Luonnossa eläimet eivät yleensä kuitenkaan kohtaa vain yhtä ympäristöongelmaa kerrallaan, vaan tavallisesti altistuvat useille ongelmille samanaikaisesti. Metallit ovat vesieläinten suurimpien ympäristöuhkien joukossa biosaatavuutensa ja pysyvyytensä takia. Eläimet pystyvät kuitenkin vastaamaan ympäristöongelmiin esimerkiksi fenotyyppisen plastisuuden kautta. Fenotyyppinen plastisuus on kyky muuttaa ilmiasua ympäristömuutoksen seurauksena. Tämä plastisuus voi myös siirtyä seuraavalle sukupolvelle, jolloin sitä kutsutaan sukupolvien väliseksi plastisuudeksi. Tässä väitöskirjatyössä tutkin laboratorio- ja kenttätöillä, miten kalat pystyvät vastaamaan vakaviin ympäristöongelmiin fenotyyppisen ja sukupolvien välisen plastisuuden kautta. Osatyössä I keskityttiin tutkimaan laboratoriossa seeprakalojen lämpöshokkiproteiinien lämpötilamuutoksen suuruudesta riippuvaa transkription ja translaation välistä suhdetta. Tutkimukset osoittivat, että transkriptio johtaa translaatioon proteiiniksi vain hyvin korkeissa lämpötiloissa, kun taas vähemmän voimakas lämpötilan nousu ei saanut aikaan kuin transkription. Työ osoitti, että kalojen molekylaarisia vasteita lämpöstressiin on tärkeä tutkia mRNA tason lisäksi myös proteiinitasolla. Osatöissä II, III ja IV lämpötilatoleranssin fenotyyppistä plastisuutta tutkittiin yksilöllisesti sekä fysiologisella että molekyylitasolla kahdella eri kalalajilla: yhdellä lauhkean ilmastovyöhykkeen lajilla (Itämeren kolmipiikki) ja yhdellä alun perin trooppisen vyöhykkeen, mutta nykyisin suurelta osin laboratorioissa kasvatetulla lajilla (seeprakala). Jokaisen yksilön lämpötilatoleranssi mitattiin tutkimuksissa ennen lämpöaalto-, kupari- ja niiden yhteisaltistusta sekä sen jälkeen. Näissä tutkimuksissa havaitsin, että lämpötilatoleranssi ja sen fenotyyppinen plastisuus olivat lajispesifisiä ja riippuivat kalan sukupuolesta. Uroskolmipiikit olivat yleisesti ottaen plastisempia kuin naaraat, kun taas seeprakaloilla naaraat olivat enimmäkseen uroksia plastisempia. Kolmipiikeillä lämpöaalto- ja kuparialtistus saivat aikaan lämpötilatoleranssin nousemisen, mikä osoittaa ympäristöstressien aiheuttavan positiivista fenotyyppistä plastisuutta. Toisaalta, vaikka lämpöaalto nosti seeprakalojen lämpötilatoleranssia, kuparialtistus ei saanut aikaan samaa vastetta. Osatyön I molekyylivastetta ei myöskään havaittu kolmipiikeillä ja ainoastaan osatyössä IV seeprakaloilla vasteet olivat samankaltaiset kuin osatyössä I osoittaen molekyylivasteiden lajispesifisyyden. Osatyössä IV tutkittiin myös ympäristömuutoksien vaikutuksia kalojen jälkeläisiin. Jälkeläisten fenotyyppiin vaikuttivat huomattavasti sekä vanhempien altistus että jälkeläisten kasvatusolosuhteet. Tämä väitöskirjatyö on tärkeä askel eteenpäin lämpötilatoleranssin tutkimisessa, sillä siinä selvitettiin sekä yksilöiden fenotyyppistä plastisuutta että mahdollista sukupolvien välisiä vaikutuksia kahden ympäristöongelman esiintyessä yhtaikaa. Tutkimuksia tehtiin myös usealla eri biologisella organisaatiotasolla lähtien molekyylivasteista adaptaation kautta tapahtuvaan evoluutioon. Tämän takia väitöskirjatyöni antaa hyvin laajan näkökulman lämpötilatoleranssin fenotyyppiseen plastisuuteen

Compromised thermal tolerance of cardiovascular capacity in upstream migrating Arctic char and brown trout—are hot summers threatening migrating salmonids?

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Are you ready for the heat? Phenotypic plasticity versus adaptation of heat tolerance in three-spined stickleback

Heat waves constitute a challenge for aquatic ectotherms. However, the thermal tolerance of animals and their individual phenotypic plasticity to respond to heat waves may be influenced by thermal history. We tested these hypotheses by comparing the upper thermal tolerance and the individual capacities of three-spined sticklebacks from populations with different thermal histories to respond to heat waves. Two populations originated from thermally polluted nuclear power plant (NPP) habitats, while four locations represented geographically adjacent control areas. To disentangle the genetic adaptation from the phenotypic plastic response, we measured the individual upper thermal tolerance and the responses at molecular level in common-garden conditions before and after a laboratory-mimicked heat wave. We found that the sticklebacks exhibit considerable phenotypic plasticity in thermal tolerance since the heat wave increased fish upper thermal tolerance significantly. The individual plasticity to respond to the heat wave was also negatively correlated with initial thermal tolerance. On the other hand, neither the thermal tolerance nor the plastic responses differed between NPP and control sites despite detection of significant but low genome-wide divergence in 10 out of 15 pairwise comparisons. Our results suggest that five decades of NPP activity with warmer water have not resulted in a detectable evolutionary change in either the upper thermal tolerance or its plasticity in three-spined sticklebacks potentially rendering them sensitive to frequent heat waves

Compromised thermal tolerance of cardiovascular capacity in upstream migrating Arctic char and brown trout - are hot summers threatening migrating salmonids?

Heat waves are threatening fish around the world, leading sometimes to mass mortality events. One crucial function of fish failing in high temperatures is oxygen delivery capacity, i.e. cardiovascular function. For anadromous salmonids, increased temperature could be especially detrimental during upstream migration since they need efficiently working oxygen delivery system in order to cross the river rapids to reach upstream areas. The migration also occurs during summer and early autumn exposing salmonids to peak water temperatures, and in shallow rivers there is little availability for thermal refuges as compared to thermally stratified coastal and lake habitats. In order to shed light on the mechanisms underpinning the capacity of migrating fish to face high environmental temperatures, we applied a physiological and molecular approach measuring cardiovascular capacities of migrating and resident Arctic char (Salvelinus alpinus) and brown trout (Salmo trutta) in Northern Norway. The maximum cardiovascular capacity of migrating fish was significantly lower compared to the resident conspecifics. The onset of cardiac impairment started only 2°C higher than river temperature, meaning that even a small increase in water temperature may already compromise cardiac function. The migrating fish were also under significant cellular stress, expressing increased level of cardiac heat shock proteins. We consider these findings highly valuable when addressing climate change effect on migrating fish and encourage taking action in riverine habitat conservation policies. The significant differences in upper thermal tolerance of resident and migrating fish could also lead changes in population dynamics, which should be taken into account in future conservation plans.</p

Copper exposure improves the upper thermal tolerance in a sex-specific manner, irrespective of fish thermal history

Ectotherms can respond to climate change via evolutionary adaptation, usually resulting in an increase of their upper thermal tolerance. But whether such adaptation influences the phenotypic plasticity of thermal tolerance when encountering further environmental stressors is not clear yet. This is crucial to understand because organisms experience multiple stressors, besides warming climate, in their natural environment and pollution is one of those. Here, we studied the phenotypic plasticity of thermal tolerance in three-spined stickleback populations inhabiting spatially replicated thermally polluted and pristine areas before and after exposing them to a sublethal concentration of copper for one week. We found that the upper thermal tolerance and its phenotypic plasticity after copper exposure did not depend on the thermal history of fish, suggesting that five decades of thermal pollution did not result in evolutionary adaptation to thermal tolerance. The upper thermal tolerance of fish was, on the other hand, increased by ∼ 1.5 °C after 1-week copper exposure in a sex-specific manner, with males having higher plasticity. To our knowledge this is the first study that shows an improvement of the upper thermal tolerance as a result of metal exposure. The results suggest that three-spined sticklebacks are having high plasticity and they are capable of surviving in a multiple-stressor scenario in the wild and that male sticklebacks seem more resilient to fluctuating environmental conditions than female.</p

Experimental copper exposure, but not heat stress, leads to elevated intraovarian thyroid hormone levels in three-spined sticklebacks (Gasterosteus aculeatus)

Climate change and pollution are some of the greatest anthropogenic threats to wild animals. Transgenerational plasticity-when parental exposure to environmental stress leads to changes in offspring phenotype-has been highlighted as a potential mechanism to respond to various environmental and anthropogenic changes across taxa. Transgenerational effects may be mediated via multiple mechanisms, such as transfer of maternal hormones to eggs/foetus. However, sources of variation in hormone transfer are poorly understood in fish, and thus the first step is to characterise whether environmental challenges alter transfer of maternal hormones to eggs. To this end, we explored the population variation and environmental variation (in response to temperature and endocrine disrupting copper) in maternal thyroid hormone (TH), transfer to offspring in a common fish model species, the three-spined stickleback (Gasterosteus aculeatus) using multiple approaches: (i) We compared ovarian TH levels among six populations across a wide geographical range in the Baltic Sea, including two populations at high water temperature areas (discharge water areas of nuclear power plants) and we experimentally exposed fish to (ii) environmentally relevant heat stress and (iii) copper for 7 days. We found that populations did not differ in intraovarian TH levels, and short-term heat stress did not influence intraovarian TH levels. However, copper exposure increased both T4 and T3 levels in ovaries. The next step would be to evaluate if such alterations would lead to changes in offspring phenotype

Combined effect of salinity and temperature on copepod reproduction and oxidative stress in brackish-water environment

Climate-induced warming and increased river inflows are forcing the Baltic Sea to radical changes in the near future; organisms living in this brackish-water ecosystem are already experiencing osmotic stress, which, together with thermal stress, may have severe consequences on the ecosystem. The aim of this work was to study the combined effect of decreasing salinity and increasing temperature on reproductive success and oxidative stress in zooplankton by using a calanoid copepod Acartia sp. as a model organism. The field study was conducted during summer 2020 in the western Gulf of Finland, using three sampling sites with naturally differing salinity levels. Additionally, the copepods from these sites were experimentally exposed to ambient or 3 degrees C elevated temperature for 72 h. The copepods derived from the deepest and the most saline sampling site suffered less oxidative damage and exhibited relatively high reproduction, while the temperature treatment itself had little effect. On the other hand, the field-based monitoring data showed otherwise; temperature increased lipid peroxidation, glutathione-s-transferase activity, or both in all three sampling sites. Meanwhile, egg production rate was negatively associated with temperature in the area with the lowest salinity. Moreover, egg production rate decreased from June to September along with increasing temperatures in the mid-salinity sampling site, while similar change occurred also in the highest-salinity site between August and September. The combined effect of salinity and sampling date on reproduction indicates the importance of even subtle salinity changes on copepods. Moreover, the data suggest that the unusually strong heatwave was responsible for increased oxidative stress during the sampling season and possibly forced a trade-off between antioxidant activity and reproductive effort.Peer reviewe

Ligand of Numb proteins LNX1p80 and LNX2 interactwith the human glycoprotein CD8a and promote itsubiquitylation and endocytosis

E3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown. In this study, we identified the human T-cell co-receptor CD8 a-chain as binding partner of the ligand of Numb proteins X1 (LNX1p80 isoform) and X2 (LNX2). Both LNX mRNAs were found expressed in T cells purified from human blood, and both proteins interacted with CD8a in human HPB-ALL T cells. By using an in vitro assay and a heterologous expression system we showed that the interaction is mediated by the PDZ (PSD95-DlgA-ZO-1) domains of LNX proteins and the cytosolic C-terminal valine motif of CD8a. Moreover, CD8a redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8a ubiquitylation, downregulation from the plasma membrane, transport to the lysosomes, and degradation. Our findings highlight the function of LNX proteins as E3 ligases and suggest a mechanism of regulation for CD8a localization at the plasma membrane by ubiquitylation and endocytosis

Increased fecal ethanol and enriched ethanol-producing gut bacteria Limosilactobacillus fermentum, Enterocloster bolteae, Mediterraneibacter gnavus and Streptococcus mutans in nonalcoholic steatohepatitis

BackgroundNon-alcoholic steatohepatitis (NASH) has become a major public health issue as one of the leading causes of liver disease and transplantation worldwide. The instrumental role of the gut microbiota is emerging but still under investigation. Endogenous ethanol (EtOH) production by gut bacteria and yeasts is an emerging putative mechanism. Microbial metagenomics and culture studies targeting enterobacteria or yeasts have been reported, but no culturomics studies have been conducted so far.AimTo assess fecal EtOH and other biochemical parameters, characterize NASH-associated dysbiosis and identify EtOH-producing gut microbes associated with the disease, fecal samples from 41 NASH patients and 24 controls were analyzed. High-performance liquid chromatography (HPLC) was used for EtOH, glucose, total proteins, triglyceride and total cholesterol. Viable bacteria were assessed with microbial culturomics. Microbial genetic material was assessed using 16S metagenomics targeting the hypervariable V3V4 region.ResultsFecal EtOH and glucose was elevated in the stools of NASH patients (p &lt; 0.05) but not triglyceride, total cholesterol or proteins. In culturomics, EtOH-producing Enterocloster bolteae and Limosilactobacillus fermentum were enriched in NASH. V3V4 16S rRNA amplicon sequencing confirmed the enrichment in EtOH-producing bacteria including L. fermentum, Mediterraneibacter gnavus and Streptococcus mutans, species previously associated with NASH and other dysbiosis-associated diseases. Strikingly, E. bolteae was identified only by culturomics. The well-known Lacticaseibacillus casei was identified in controls but never isolated in patients with NASH (p &lt; 0.05).ConclusionElevated fecal EtOH and glucose is a feature of NASH. Several different EtOH-producing gut bacteria may play an instrumental role in the disease. Culturomics and metagenomics, two complementary methods, will be critical to identify EtOH-producing bacteria for future diagnostic markers and therapeutic targets for NASH. Suppression of EtOH-producing gut microbes and L. casei administration are options to be tested in NASH treatment

Are you ready for the heat? Phenotypic plasticity versus adaptation of heat tolerance in three-spined stickleback

Heat waves constitute a challenge for aquatic ectotherms. However, the thermal tolerance of animals and their individual phenotypic plasticity to respond to heat waves may be influenced by thermal history. We tested these hypotheses by comparing the upper thermal tolerance and the individual capacities of three-spined sticklebacks from populations with different thermal histories to respond to heat waves. Two populations originated from thermally polluted nuclear power plant (NPP) habitats, while four locations represented geographically adjacent control areas. To disentangle the genetic adaptation from the phenotypic plastic response, we measured the individual upper thermal tolerance and the responses at molecular level in common-garden conditions before and after a laboratory-mimicked heat wave. We found that the sticklebacks exhibit considerable phenotypic plasticity in thermal tolerance since the heat wave increased fish upper thermal tolerance significantly. The individual plasticity to respond to the heat wave was also negatively correlated with initial thermal tolerance. On the other hand, neither the thermal tolerance nor the plastic responses differed between NPP and control sites despite detection of significant but low genome-wide divergence in 10 out of 15 pairwise comparisons. Our results suggest that five decades of NPP activity with warmer water have not resulted in a detectable evolutionary change in either the upper thermal tolerance or its plasticity in three-spined sticklebacks potentially rendering them sensitive to frequent heat waves.</p