Primary cultured gill models are useful for investigating fish gill epithelium barrier properties. Typically, gill cell culture methods utilize commercially available heterologous serum supplements rather than native serum. This is because heterologous serum is easy to obtain and facilitates the development of models that mimic the gill very well. Also, native serum (or plasma) has historically had mixed success in primary cultured fish epithelium models. This study examined the effects of the blood anti-coagulating agent heparin and native serum on the barrier properties of a primary cultured trout (Oncorhynchus mykiss) gill model. Development of epithelia in the presence of heparin decreased transepithelial electrical resistance (TER) and increased paracellular movement of [3H]polyethylene glycol (MW 400 Da, PEG-400). In association, transcript abundance of genes encoding tight junction (TJ) proteins tricellulin, zonula occludens-1, cingulin, and claudin (cldn) -1, -8b, -8c, -8d, -12, -23a, -27b, -28b, and -29a decreased, while cldn30 and -32a mRNA abundance increased. Acute treatment with heparin for 24 h had a qualitatively similar impact on TER and paracellular permeability, but a less pronounced impact on TJ-associated genes. In contrast, replacing 10
Heat loss due to a large surface-area-to-volume ratio makes surviving periods of cold temperatures a challenge for small endothermic animals. Many temperate, non-migratory birds survive cold winter nights by increasing heat production through shivering, maintaining their body temperature at the cost of the energy provided by fat stores. An additional strategy may be the use of facultative hypothermia, allowing body temperature to decrease overnight to conserve energy rather than maintaining normothermic body temperature. In this study, we examined the relationship between overnight body temperature and fat loss under a cold temperature challenge in black-capped chickadees (Poecile atricapillus). To investigate this, we brought 10 wild black-capped chickadees into captivity and used quantitative magnetic resonance analysis and temperature sensitive radio transmitters to compare changes within-individual overnight fat loss and body temperature at air temperature of 20 °C versus 3 °C. Most of the black-capped chickadees showed both reduced body temperature and greater fat loss at 3 °C compared to 20 °C. Furthermore, individuals exhibiting greater decreases in body temperature showed lower overnight fat loss. Interestingly, some individuals did not drop their body temperature across the air temperature conditions. These results suggest individual variation in use of facultative hypothermia to reduce fat loss overnight and provide valuable insights into the metabolic strategies small, non-migratory birds use to survive cold nights.
Increasing thermal variability, including sudden cold snaps, is a predicted consequence of climate change that may require endothermic homeotherms to make rapid physiological adjustments to maintain thermal homeostasis. Birds exhibit pronounced seasonal phenotypic changes, yet the cellular mechanisms underlying these responses remain poorly understood. Because shivering thermogenesis is central to avian heat production, skeletal muscle ultrastructure changes seasonally to support thermogenic performance. We acclimated summer-phenotype house sparrows (Passer domesticus) to 25 °C (control/constant warm), 3 °C (stable cold), and mean 3 °C (fluctuating cold) conditions for six weeks and quantified pectoralis muscle fiber diameter, number of nuclei per mm of fiber, and myonuclear domain (MND). We also measured expression of the muscle-associated microRNA miR-1 and its target gene Itm2a, which regulate muscle hypertrophy in mammals. Thermal acclimation did not affect muscle ultrastructure or gene expression, suggesting that summer-phenotype sparrows are well equipped to tolerate variable thermal conditions. In contrast, comparisons with winter-phenotype birds from a previous study revealed clear seasonal differences. Summer-phenotype birds had larger diameter muscle fibers, fewer myonuclei per mm of fiber, and larger MNDs than winter-phenotype birds across all acclimation treatments. Similarly, miR-1 expression was lower and Itm2a expression higher in summer- than winter-phenotype birds, consistent with their roles in regulating muscle hypertrophy. These findings indicate that seasonal shifts, rather than short-term thermal variability, drive changes in pectoralis muscle ultrastructure and associated molecular regulators, highlighting seasonal remodelling as an important mechanism supporting thermogenic physiology in house sparrows.
The pirarucu Arapaima gigas is an obligate air-breathing fish with a massive internal air-breathing organ, yet it also retains substantial gills. In this study, Arapaima were exercised for 10 min either in water or in air, or were exposed to air for one hour. During subsequent recovery from exercise or air exposure aerial and aquatic oxygen uptake and ventilatory activity were recorded for two hours to assess the contribution of aerial respiration to repayment of an oxygen debt. Compared to the initial control period, oxygen uptake (MO2) was almost doubled during air exposure. A doubling of total MO2 was also recorded during the first 30 min of recovery in all three experimental series. Thereafter MO2 returned to resting values, indicating a quick recovery from exercise as well as from air exposure. Aerial and aquatic ventilation were significantly elevated during initial recovery from all treatments. Air-breathing frequency returned to resting values more quickly after air exposure than after exercise in water or air. Relating oxygen uptake from air and water to ventilatory activity revealed that breathing air was about 200-fold more effective than breathing water. Aerial oxygen uptake contributed between 69
Christmas Island in the Indian Ocean hosts large populations of land crabs, including the gecarcinids Gecarcoidea natalis and Tuerkayana celeste and the grapsid Geograpsis grayi. All three have transitioned into terrestrial habitats via marine environments, but through different evolutionary routes, and with different life history traits. These species provide an opportunity to investigate the incorporation of chitin, protein and minerals (particularly calcium and magnesium carbonates) into the arthropod exoskeleton given their shared terrestrial transition, but different evolutionary histories and contrasting habitat use. While these minerals are known to provide mechanical strength and resilience to external pressure, little is known of interspecific differences. If proportions are constrained by phylogeny we predict the gecarcinids would be more similar to one another than to the grapsid, but if there has been ecological specialisation this would not necessarily be the case. While there was more chitin present in the exoskeleton of the grapsid compared to the geocarcinids, no further significant differences in protein or mineral composition were detected. The [Ca2+]: [Mg2+] ratio differed significantly between all three species, but was lowest in the two most terrestrial species (G. natalis, T. celeste and G. grayi exoskeleton [Ca2+]: [Mg2+]: 9.25 (± 0.17), 19.9 (± 1.89), 15.6 (± 0.35), respectively) and not the two most closely related. It appears that higher [Mg2+] is associated with increased terrestriality, reflecting the chemical and physical properties associated with higher [Mg2+] in the mineral component of the crustacean exoskeleton, and provides limited support for any phylogenetically driven differences, perhaps with the exception of chitin.
The maintenance of body temperature (Tb) and hydration state within tolerable ranges is crucial for effective physiological functioning and thus survival. It is well established that deviations in Tb and hydration state can have immediate effects on organismal health, and recent work has demonstrated that, despite surviving the initial impacts of such challenges, individuals may experience profound long-term impacts on fitness, though the mechanisms driving these long-term impacts remain unclear. One potential mechanism underlying detrimental long-term effects is oxidative stress, yet the relationships among increased Tb, dehydration, and oxidative stress dynamics, as well as whether these relationships can be altered through adaptation, are mostly unexplored. Accordingly, we conducted complementary field and lab experiments to investigate seasonal, behavioral, thermal, and hydric effects on oxidative stress in two species of arid-adapted Crotalus rattlesnakes representing three populations from two xeric locations with differing aridity. Regardless of experiment or treatment, we found that C. pyrrhus, the most abundant species in the more arid location, had consistently lower reactive oxygen metabolite (ROM) concentrations than did C. atrox from the same and the less arid locations. Furthermore, the C. atrox population from the more arid site had ROM concentrations intermediate and often significantly different from the other two populations. Interestingly, when Tb and activity were similar between the C. atrox populations (e.g., in Spring), ROM concentrations were similar. Contrary to our hypotheses, we found no consistent patterns relating antioxidant capacity to any environmental or phylogenetic variable. Lastly, hydration state did not influence either ROM concentration or antioxidant capacity. Overall, our study demonstrates that while water deprivation and increased temperatures don’t necessarily induce oxidative stress in species tolerant of these conditions, results are consistent with the hypothesis that species that are more specialized for extremely arid environments have reduced ROM concentrations. Such insight is critical as we attempt to understand how species have adapted to arid conditions and how species may respond to a warming and drying globe.
In invertebrates, hemocytes are the principal effector cells of innate immunity, and a growing body of evidence indicates that the circulation of immune cells is under circadian and nycthemeral control across both vertebrate and invertebrate taxa. Yet whether such temporal organisation extends to terrestrial gastropods, increasingly used as comparative models in physiology and ecotoxicology through hemocyte-based biomarkers, remains poorly documented. This study investigated nycthemeral variations in hemocyte-related parameters in juvenile, sub-adult and adult Cornu aspersum maintained under controlled laboratory conditions. Snails were sampled at four time points over a 24 h cycle (06:00, 12:00, 18:00 and 24:00; n = 20 per developmental stage and sampling time). Circulating hemocyte concentration, hemocyte viability, proportions of hemocyte types I and II, and micronucleus frequency were assessed following standardised procedures. Significant temporal variations were observed in circulating hemocyte concentration, with lower values generally recorded during evening and nocturnal sampling periods (18:00 and 24:00). Similar nycthemeral patterns were observed across developmental stages, although they were less pronounced in juveniles. In contrast, hemocyte viability, hemocyte profile distribution and micronucleus frequency remained comparatively stable throughout the 24 h cycle, suggesting the absence of marked cytogenomic fluctuations associated with sampling time under the tested conditions. These findings indicate that nycthemeral physiological rhythms may substantially influence hemocyte circulation dynamics and hemolymph accessibility in terrestrial gastropods, while exerting limited effects on basal cellular integrity. The study highlights the importance of temporal standardisation in hemocyte-based biomarker protocols and supports the integration of chronobiological considerations into ecotoxicological and comparative physiological investigations involving terrestrial molluscs.
The moult represents an important seasonal change in the physiology of phocid seals. However, physiological measurements are challenging due to the impracticalities of instrumenting moulting animals. The aim of this study was (i) to assess the feasibility of a long-term deployment of fully implantable loggers to measure heart rate (HR) and sub-blubber temperature (Tb) in harbour seals (Phoca vitulina) and (ii) to compare HR and Tb during moult and post-moult periods. Loggers were surgically implanted on an adult and sub-adult harbour seal in captivity. Animals healed quickly from surgery and loggers provided 65 days of continuous data before removal of the device. Results showed that in the adult when hauled out and in the water HR was lower during the moult compared with the post-moult. For the sub-adult, HR while in the water was also higher during the post-moult, however, when hauled out there was no difference in HR between the moult and post-moult periods. When hauled out, Tb was greater for both animals during the moult compared with the post-moult. During haulout events, Tb increased over time in both periods and Tb in both animals was lower during post moult compared to the moult period. In the sub-adult, Tb decreased with time in the water during the moult but showed little change during the post-moult period, whereas no temporal change was evident in the adult during either period. This study demonstrated that fully implantable loggers are a useful research tool allowing measurement of physiological parameters that are otherwise difficult to measure in pinnipeds. Physiological measurements suggested that the requirement to maintain higher skin temperature for hair growth during the moult was offset by lower metabolism to save energy in water, and on land in the case of adults. However, a decrease in body temperature with time in water in the sub-adult indicated that a smaller body size and less well-developed blubber layer is not as effective in reducing heat loss compared to adults. Future physio-logging on pinnipeds throughout their annual lifecycle may therefore benefit from similar techniques used to implant loggers.
This experiment was conducted to evaluate the effects of dietary nanocurcumin on growth performance, feed utilization, innate immunity, antioxidant capacity, biochemical indices, and air exposure stress resistance in European seabass (Dicentrarchus labrax) fingerlings. Five isonitrogenous (47
Electrophysiology is an emerging approach in the context of environmental toxicology, where it represents a non-invasive biomarker that has been little studied in tropical fish. In this study, we evaluated electrocardiographic (ECG) and electromyographic (EMG) responses in Hyphessobrycon heterorhabdus, a native Amazonian species considered a promising biological model for ecotoxicological studies, and Menthol was selected as the pharmacological model for methodological validation in this species. Fish were exposed to 30 mg L− 1 for three minutes and subsequently transferred to clean water to assess recovery. The ECG showed a reduction in heart rate and an increase in the wave intervals in fish exposed to menthol. The EMG recorded the occurrence of transient muscle spasms before a progressive decrease in contraction strength as anesthesia deepened. After recovery, both ECG and EMG tracings returned to baseline conditions, indicating the reversibility of menthol-induced anesthesia. Thus, electrophysiological markers in H. heterorhabdus proved to be effective tools for detecting short-term physiological changes and supports their application in studies involving environmental stressors or chemical exposures.
Pacific lamprey, an anadromous fish native to the North Pacific Ocean, are imperiled in parts of their range. Many aspects of their biology are not fully understood, including the physiology of their spawning migration. Pacific lamprey exhibit two divergent reproductive life histories: early-maturing and late-maturing. Interior Columbia River early-maturing lamprey complete the final stages of gonadal maturation 10 months after entering freshwater. From the same migrating cohort, the late-maturing lamprey wait 22 months to complete maturation. This study sought to characterize the reproductive phenotypes of these two Pacific lamprey life histories. Migrating adult Pacific lamprey that had entered fresh water were collected and held for close to two years to monitor changes in morphology and physiology. The lamprey in this study, of approximately equal sex ratio, clearly separated into early- or late-maturing life histories based on differences in timing of reproductive development, morphology of length, weight, interdorsal fin gap, and plasma concentrations of estradiol-17β (E2), but not plasma androstenedione. Average plasma E2 levels in females and males were significantly higher in the early-maturing fish compared to the late-maturing fish by November, nearly half a year before any morphological divergence. This novel physiological indicator could benefit restoration activities (translocation and artificial propagation) by providing advanced identification of the two maturation life histories present in the same migration cohort. In conclusion, this study provides several morphological and physiological features that can be used to identify early- and late-maturing Pacific lamprey within the 10 month period prior to when the early-maturing fish mature.
Ultraviolet-B (UVB) radiation is a critical environmental stressor that affects aquatic organisms, particularly during vulnerable early life stages. This study investigated the effects of low ambient UVB exposure (1.0 W/m2, corresponding to 6–12
Temperature is a decisive factor for life on earth because chemical reaction speed increases with temperature. Insects are ectothermic at rest, and in this way are subject to the direct influence of environmental temperature on growth, development and reproduction. Thermal performance curves (thermal reaction norms) describe the change of metabolism and development speed with temperature, which increase with temperature differently in different species and developmental stages. This review summarizes the main principles and findings on these relationships in insects, provides hints on how to measure respiratory performance curves, and reports different attempts to describe them mathematically. In addition, it summarizes some behavioral and physiological strategies insects have developed to escape the limitations of their thermal performance relationships.
Synchronization to the environmental day-night cycle is the key to survival in many animals. For a photoinduced physiological response to occur, Critical Daylength (CD) is required for that species. Some studies have shown changes in pectoral muscles and adipose tissue of migratory birds under the influence of 'Continuous light Pulse' (CP) photoperiodic schedules, but data under the influence of 'Skeleton Photoperiod' (SKP) is still lacking. Two groups of Redheaded Buntings were exposed to SKPs along with one group on shortday control, to test their effects on histological changes via histomorphometric analysis of pectoral muscle fibers and adipose tissue. The SKP groups were designed such that one tended to be photosensitive, while the other tended to be photostimulatory. The tissues of interest were cryosectioned and stained with either haematoxylin-eosin or oil red-O (ORO) stain. Thereafter, microscopy and imaging were done. We measured pectoral muscle fiber thickness, neutral lipid droplet size in muscle fibers, adipocyte area, adipocyte number density/ 1000 µm2, and adipocyte area frequency. Significant differences were observed in the morphometry of muscle fibers and adipocyte parameters of these groups. Depending on the perceived daylengths, positive correlations were observed between the characteristics of these tissues. Previous studies on buntings demonstrated physiological changes under such light regimes, but here we present evidence at the histological levels through histomorphometric analysis. It is suggested that adipocyte fat storage precedes muscle hypertrophy. The results raise a possibility for involvement of endocrine factors between pectoral muscles and adipose tissue in this migratory bird.
Tambaqui (Colossoma macropomum) is a fruit and seed-consuming omnivorous frugivorous fish. It has the metabolic capacity to process dietary carbohydrates, representing an interesting biological model for metabolic studies. We aimed to evaluate the glycaemic response and intermediate metabolism of tambaqui after administering different carbohydrates. A total of 300 juvenile tambaqui with an average weight of 111.5 ± 49.8 g were used. The fish were orally administered 200 mg/100 g body weight of glucose, fructose, sucrose, starch, or saline solution (control). Blood and liver samples were collected at 0 h (baseline) and 3, 6, 9, and 12 h after administration. Plasma glucose concentration, blood metabolites, and the hepatic activity of key metabolic enzymes were analysed. Elevated plasma glucose concentrations were observed 3 h after glucose, fructose, and sucrose administration and 6 h after starch administration. After 9 h, blood glucose levels in all groups returned to baseline. Increased hepatic activity of pyruvate kinase and glucokinase was observed 9 h after the administration of all the tested sugars. This was associated with a decrease in the activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase. Fish fed starch and glucose exhibited increased hepatic fatty acid synthase activity at 6 and 9 h, respectively. Tambaquis have a high capacity to use all the tested sugars for energy purposes. However, fructose and sucrose did not stimulate metabolic synthesis of lipids because of excess sugar intake.
Historic declines of American black duck (Anas rubripes, hereafter black duck) and lesser scaup (Aythya affinis) populations may be the result of multiple factors including resource availability on migration and wintering landscapes. Bioenergetics modeling is used to calculate the carrying capacity of a landscape by estimating energy demand and energy supply. Past estimates of resting metabolic rate (RMR) and behavioral specific multipliers to RMR, physiologic parameters required for calculating energetic demand, are limited and, in some cases, based upon dated past research. We used open-flow respirometry techniques to estimate RMR and behavioral specific multipliers to RMR for captive black ducks (n = 6) and lesser scaup (n = 6) at U.S. Geological Survey’s Patuxent Wildlife Research Center in Laurel, Maryland during winter 2015/2016. RMR estimates for black ducks (357.28 kJ/bird/day) and lesser scaup (381.43 kJ/bird/day) in this study fell within the range of previously published estimates for each species. Behavioral multiplier estimates were different between species and from those reported in previous studies. We found that similarities between multiplier estimates for certain behaviors in each species justified combining behaviors into homogenous groups representing high, medium, low, and extreme low (or RMR) energy behavior classes. We tested the effects of six covariates (time of day [AM/PM], presence of live or decoy cohort, food presence [Y/N], weight [g], air temperature [C], and water temperature [C]) on energy expenditure within each behavioral grouping and within each species. Black ducks engaged in “medium” and “high-energy behaviors” experienced increasing energetic demand as water temperature decreased, and energetic demand interactively increased with higher energy behaviors. Energy expenditure increased with increasing mass in both black ducks and lesser scaup engaged in “high-energy behaviors.” Lesser scaup engaged in “medium-energy behaviors” expended more energy in the morning than in the afternoon. Lesser scaup engaged in “high-energy behaviors” expended more energy when food was absent than when present. Results from this study technologically improve past estimates, refine estimates derived using allometric equations, simplify the underlying calculations required for bioenergetics modeling and can help improve the accuracy of landscape carrying capacity estimates resulting from those models.
Global ecosystems are under constant threat from nonindigenous species, with the potential to cause both economic and ecological harm on a global scale. The green crab (C. maenas) has invaded global coastlines, increasing resource competition and predating upon native organisms. Recent evidence also suggests the green crabs within the Canadian Pacific estuaries possess an acute tolerance to extremely dilute and freshwater (FW) environments compared to historical literature. If Pacific green crabs are indeed tolerant to limited FW exposure, this could increase their ecological range to forage or avoid competitive interactions and predation. Here we evaluate the response of Pacific C. maenas to acute FW exposure, and the implications of increasing temperatures on haemolymph composition in a dilute environment. With an average survival time of 25.4 ± 1.22 h in FW following acclimation to 6.6 ppt seawater (SW), Pacific green crabs exhibited an acute tolerance to exposure in extremely dilute environments. Pacific green crab’s haemolymph osmolality, branchial cyclic AMP (cAMP), oxygen consumption, haemolymph protein, and hemocyanin are largely unaffected within at least the first 6 h of FW exposure. Branchial cAMP in particular was shown to be over 2-fold higher in green crabs acclimated to 18 °C when compared to those acclimated at 12 °C, suggesting that endocrine differences might be correlated to temperature rather than salinity. We propose that the invasive green crabs residing within the Canadian Pacific estuaries of Vancouver Island exhibit a limited but increased tolerance to extremely dilute environments compared to those historically measured in Atlantic populations.
Total ammonia nitrogen (TAN) is a major cellular stressor in intensive Macrobrachium rosenbergii aquaculture; however, the molecular mechanisms underlying hepatopancreatic responses to chronic ammonia exposure remain insufficiently understood. In this study, juvenile prawns were exposed for seven days to TAN concentrations of 0 mg L⁻¹ (control), 2 mg L⁻¹, and 50 mg L⁻¹ under stable environmental conditions (27–28 °C, pH 7.5–8.0, dissolved oxygen 5–6 mg L⁻¹). No mortality was observed in the control group (100.0 ± 0.0
In 1996, based on the ideas of Max Kleiber, we proposed a respirometric method in this journal for the quantitative determination of the roles of protein, lipid and carbohydrate as substrates for fueling aerobic metabolism in fish on an instantaneous basis. Here I provide a 30-year retrospective on its performance, explaining how it works, methodological challenges, the applications for which it has been used, its strengths and potential flaws, and the important issues to be addressed going forward. The approach is based on the simultaneous measurement of the rates of O2 consumption (ṀO2), CO2 excretion (ṀCO2) and N-waste excretion (ṀN = Ṁamm + Ṁurea−N) under steady-state conditions in the whole fish when anaerobic metabolism is not occurring. These allow the calculation of the Respiratory Quotient (RQ = ṀCO2/ṀO2) and the Nitrogen Quotient (NQ = ṀN /ṀO2), and from these, the fractional contributions of each of the three fuels. Principal methodological challenges arise from the difficulties of measuring ṀCO2, and to a lesser extent ṀN, in water. To date, the approach has been used mainly to study fuel use during feeding, fasting, starvation, sustainable exercise, and at different temperatures. In general, lipid and carbohydrates have emerged as the major fuels burned in ammoniotelic fish (where ammonia is the predominant N-waste product), while protein is conserved, though protein metabolism may be more important in ureotelic fish (where urea-N is the predominant N-waste product) and air-breathers. Possible unidentified N- products of protein oxidation, the action of the anaerobic gut microbiome in generating ṀN in the absence of ṀO2, and the ability of the gill microbiome to convert N-waste to di-nitrogen (N2) are highlighted as potential flaws.
Torpor is a reversible hypometabolic state marked by decreases in body temperature and energy expenditure. Leveraging advanced genetic and physiological techniques, recent studies have begun to identify hypothalamic and brainstem neurons that initiate and maintain this state. These tools nevertheless come with substantial limitations, and this manuscript discusses key interpretational caveats and practical considerations. Looking ahead, a central challenge is to test whether multi-day hibernation arises from retuning of the same core circuits that govern daily torpor.
