Evolutionary Adaptation of Protein Turnover in White Muscle of Stenothermal Antarctic Fish: Elevated Cold Compensation at Reduced Thermal Responsiveness.

Protein turnover is highly energy consuming and overall relates to an organism's growth performance varying largely between species, e.g., due to pre-adaptation to environmental characteristics such as temperature. Here, we determined protein synthesis rates and capacity of protein degradation in white muscle of the cold stenothermal Antarctic eelpout (Pachycara brachycephalum) and its closely related temperate counterpart, the eurythermal common eelpout (Zoarces viviparus). Both species were exposed to acute warming (P. brachycephalum, 0 °C + 2 °C day-1; Z. viviparus, 4 °C + 3 °C day-1). The in vivo protein synthesis rate (Ks) was monitored after injection of 13C-phenylalanine, and protein degradation capacity was quantified by measuring the activity of cathepsin D in vitro. Untargeted metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy was used to identify the metabolic processes involved. Independent of temperature, the protein synthesis rate was higher in P. brachycephalum (Ks = 0.38-0.614 % day-1) than in Z. viviparus (Ks= 0.148-0.379% day-1). Whereas protein synthesis remained unaffected by temperature in the Antarctic species, protein synthesis in Z. viviparus increased to near the thermal optimum (16 °C) and tended to fall at higher temperatures. Most strikingly, capacities for protein degradation were about ten times higher in the Antarctic compared to the temperate species. These differences are mirrored in the metabolic profiles, with significantly higher levels of complex and essential amino acids in the free cytosolic pool of the Antarctic congener. Together, the results clearly indicate a highly cold-compensated protein turnover in the Antarctic eelpout compared to its temperate confamilial. Constant versus variable environments are mirrored in rigid versus plastic functional responses of the protein synthesis machinery.

Protein Synthesis Determined from Non-Radioactive Phenylalanine Incorporated by Antarctic Fish.

Direct measurements of temperature-dependent weight gains are experimentally challenging and time-consuming in long-lived/slow-growing organisms such as Antarctic fish. Here, we reassess methodology to quantify the in vivo protein synthesis rate from amino acids, as a key component of growth. We tested whether it is possible to avoid hazardous radioactive materials and whether the analytical pathway chosen is robust against analytical errors. In the eelpout, Pachycara brachycephalum, 13C9H1115N1O2 phenylalanine was injected intraperitoneally and muscle tissue was sampled before injection and at 1.5 h time intervals up to 6 h thereafter. The incorporation of 13C15N-labeled-phenylalanine into muscle was monitored by quantification of bound and free phenylalanine through liquid chromatography-mass spectrometry. We found an increase in the pool of labeled, free phenylalanine in the cytosolic fraction that leveled off after 4.5 h. The labeled phenylalanine bound in the proteins increased linearly over time. The resulting protein synthesis rate (Ks) for P. brachycephalum was as low as 0.049 ± 0.021% day-1. This value and its variability were in good agreement with literature data obtained from studies using radioactive labels, indicating that this methodology is well suited for characterizing growth in polar fish under in situ conditions in remote areas or on research vessels.

Long-term effects of elevated pCO2 levels on the expression of Chaoborus-induced defences in Daphnia pulex.

Increased carbon dioxide from fossil fuel combustion results in an enrichment of CO2 in the global carbon cycle. Recent evidence indicates that rising atmospheric CO2 impacts the partial pressure of carbon dioxide (pCO2) in freshwaters. This affects freshwater biota by disrupting chemical communication between predator and prey. One such well-described predator-prey interaction is the phantom midge larva Chaoborus preying on the freshwater crustacean Daphnia pulex. To counter Chaoborus predation, D. pulex develops defensive neckteeth in response to chemical cues. The strength of neckteeth expression is reduced when D. pulex experience elevated pCO2 levels. This is discussed to directly impair predator perception and results in reduced defence expression. However, it is not known whether there are also long-term effects associated with continuous elevated pCO2. Here, we investigated the effect of long-term exposure of D. pulex to elevated pCO2 levels in a life-table experiment over three generations. Using a flow-through system, we continuously exposed D. pulex to cues released by the predatory larva Chaoborus and control or elevated pCO2 levels. We determined morphological defence expression in the 2nd juvenile instar and the number of neonates as a measure for life-history traits over three successive generations. We detected that elevated pCO2 significantly reduces the expression of predator-induced morphological defences (i.e. neckteeth) and life-history parameters (i.e. number of neonates) in successive generations. Our data clearly show that at least three generations become more vulnerable to predation without indications of transgenerational acclimation. As Daphnia is a keystone grazer of freshwater ecosystems, this may destabilise population growth rates. In conclusion, long-term effects of pCO2-induced reduction of predator-induced plasticity may significantly affect trophic interactions.

Elevated pCO2 affects behavioural patterns and mechano-sensation in predatory phantom midge larvae Chaoborus obscuripes.

Aquatic acidification is a major consequence of fossil fuel combustion. In marine ecosystems it was shown, that increasing pCO2 levels significantly affect behavioural and sensory capacities in a diversity of species. This can result in altered predator and prey interactions and thereby change community structures. Just recently also CO2 dependent acidification of freshwater habitats has been shown. Also here, increased levels of pCO2 change organisms' behaviour and sensory capacities. For example, the freshwater crustacean Daphnia's ability to detect predators and accurately develop morphological defences was significantly reduced, rendering Daphnia more susceptible to predation. It was speculated that this may have cascading effects on freshwater food webs. However, for a comprehensive understanding of how increased levels of CO2 affect trophic interactions, it is also important to study how CO2 affects predators. We tested this using the dipeteran phantom midge larva Chaoborus obscuripes, which is a world-wide abundant inhabitant of freshwater impoundments. We monitored activity parameters, predation parameters, and predation rate. Chaoborus larvae are affected by increased levels of pCO2 as we observed an increase in undirected movements and at the same time, reduced sensory abilities to detect prey items. This is likely to affect the larvae's energy budgets. Chaoborus is a central component of many freshwater food-webs. Therefore, CO2 effects on predator and prey levels will likely have consequences for community structures.

Mitotic activity patterns and cytoskeletal changes throughout the progression of diapause developmental program in Daphnia.

BACKGROUND: Diapause is a form of dormancy that is genetically predetermined to allow animals to overcome harsh environmental conditions. It is induced by predictive environmental cues bringing cellular activity levels into a state of suspended animation. Entering diapause requires organismal, molecular and cellular adaptation to severely reduced energy flows. Cells must therefore have evolved strategies that prepare them for periods with limited metabolic resources. However, changes that occur on the (sub-)cellular level have not been thoroughly described. RESULTS: We investigated mitotic activity and we monitored cytoskeletal network changes in successive stages of diapausing and non-diapausing Daphnia magna embryos using (immuno-)fluorescent labeling. We find that embryos destined to diapause show a delayed and 2.5x slower mitotic activity in comparison to continuously developing embryos. Development is halted when D. magna embryos reach ~ 3500 cells, whereupon mitotic activity is absent and cytoskeletal components are severely reduced, rendering diapause cells compact and condensed. CONCLUSION: In the initiation phase of diapause, the slower cell division rate points to prolonged interphase duration, preparing the cells for diapause maintenance. During diapause, cytoskeletal depletion and cellular condensation may be a means to save energy resources. Our data provide insights into the sub-cellular change of diapause in Daphnia.