Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology最新文献

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g., genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for “non-molecular” physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest.

The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.

Exposure to environmental changes often results in the production of reactive oxygen species (ROS), which, if uncontrolled, leads to loss of cellular homeostasis and oxidative distress. However, at physiological levels these same ROS are known to be key players in cellular signaling and the regulation of key biological activities (oxidative eustress). While ROS are known to mediate salinity tolerance in plants, little is known for the animal kingdom. In this study, we use the Mediterranean crab Carcinus aestuarii, highly tolerant to salinity changes in its environment, as a model to test the healthy or pathological role of ROS due to exposure to diluted seawater (dSW). Crabs were injected either with an antioxidant [N-acetylcysteine (NAC), 150 mg·kg⁻¹] or phosphate buffered saline (PBS). One hour after the first injection, animals were either maintained in seawater (SW) or transferred to dSW and injections were carried out at 12-h intervals. After ≈48 h of salinity change, all animals were sacrificed and gills dissected for analysis. NAC injections successfully inhibited ROS formation occurring due to dSW transfer. However, this induced 55% crab mortality, as well as an inhibition of the enhanced catalase defenses and mitochondrial biogenesis that occur with decreased salinity. Crab osmoregulatory capacity under dSW condition was not affected by NAC, although it induced in anterior (non-osmoregulatory) gills a 146-fold increase in Na⁺/K⁺/2Cl⁻ expression levels, reaching values typically observed in osmoregulatory tissues. We discuss how ROS influences the physiology of anterior and posterior gills, which have two different physiological functions and strategies during hyper-osmoregulation in dSW.

We explored the relationship between gestational states, fecundity, and steroid hormone levels in three species of live-bearing fish with different maternal provisioning strategies. We studied two lecithotrophic species, Gambusia affinis and Xiphophorus couchianus, where embryos feed exclusively on yolk stored in the eggs, and one matrotrophic species, Heterandria formosa, which actively transfers nutrients to embryos through a follicular placenta. We measured water-borne cortisol, estradiol, and progesterone along with brood size (fecundity) and gestational stage(s). We examined the physiological costs of both maternal provisioning modes. Matrotrophy likely imposes energetic demands due to active nutrient transfer, while lecithotrophy may incur costs from carrying many large embryos. We hypothesized that fecundity, gestational stage, and hormones would covary differently in lecithotrophic vs. matrotrophic species. We found no relationships between hormones and fecundity or gestational stage in any species. However, in H. formosa, we found a positive relationship between estradiol levels and female mass, and a negative relationship between progesterone levels and female mass indicating a change in the circulating levels of both hormones as females grow. We observed differences in average hormone levels among species: the matrotrophic species had higher progesterone and lower estradiol compared to lecithotrophic species. Higher estradiol in lecithotrophic species may relate to egg yolk formation, while placental structures could play a role in progesterone production in matrotrophic species. Elevated cortisol in H. formosa suggests either higher energetic costs or a preparative role for reproduction. Our findings highlight progesterone's importance in maintaining gestation in matrotrophic species, like other placental species.

Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na⁺, K⁺)-ATPase and the apical V(H⁺)-ATPase. The kinetic characteristic of gill (Na⁺, K⁺)-ATPase and the mRNA expression of its α subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod α subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (∼94% identity) compared to the β-subunit (∼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-β interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na⁺, K⁺)-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence.

The predicted global warming of surface waters can be challenging to aquatic ectotherms like freshwater mussels. Especially animals in northern temperate latitudes may face and physiologically acclimate to significant stress from seasonal temperature fluctuations. Na⁺/K⁺-ATPase enzyme is one of the key mechanisms that allow mussels to cope with changing water temperatures. This enzyme plays a major role in osmoregulation, energy control, ion balance, metabolite transport and electrical excitability. Here, we experimentally studied the effects of temperature on Na⁺/K⁺-ATPase activity of gills in two freshwater mussel species, Anodonta anatina and Unio tumidus. The study animals were acclimated to three ambient temperatures (+4, +14, +24 °C) and Na⁺/K⁺-ATPase activity was measured at those temperatures for each acclimation group. Both species had their highest gill Na⁺/K⁺-ATPase activity at the highest acclimation temperature. Na⁺/K⁺-ATPase activity of gills exhibited species-specific differences, and was higher in A. anatina than U. tumidus in all test groups at all test temperatures. Temperature dependence of Na⁺/K⁺-ATPase was confirmed in both species, being highest at temperatures between +4 and + 14 °C when Q₁₀ values in the acclimation groups varied between 5.06 and 6.71. Our results underline the importance of Na⁺/K⁺-ATPase of gills for the freshwater mussels in warming waters. Because Na⁺/K⁺-ATPase is the driving force behind ciliary motion, our results also suggest that in warming waters A. anatina may be more tolerant at sustaining vigorous ciliary action (associated with elevated respiration rates and filter-feeding) than U. tumidus. Overall, our results indicate great flexibility of the mussel's ecophysiological characteristics as response to changing conditions.

Light detection underlies a variety of animal behaviors, including those related to spatial orientation, feeding, avoidance of predators, and reproduction. Ctenophores are likely the oldest animal group in which light sensitivity based on opsins evolved, so they may still have the ancestral molecular mechanisms for photoreception. However, knowledge about ctenophore photosensitivity, associated morphological structures, molecular mechanisms involved, and behavioral reactions is limited and fragmented. We present the initial experiments on the responses of adult Beroe ovata to high-intensity light exposure with different spectra and photosensitivity in various parts of the animal's body. Ctenophores have shown a consistent behavioral response when their aboral organ is exposed to a household-grade laser in the violet spectrum. To investigate the genes responsible for the photosensitivity of Beroidae, we have analyzed transcriptome and genome-wide datasets. We identified three opsins in Beroe that are homologous to those found in Mnemiopsis leidyi (Lobata) and Pleurobrachia bachei (Cydippida). These opsins form clades Ctenopsin1, 2, and 3, respectively. Ctenopsin3 is significantly distinct from other ctenophore opsins and clustered outside the main animal opsin groups. The Ctenopsin1 and Ctenopsin2 groups are sister clusters within the canonical animal opsin tree. These two groups could have originated from gene duplication in the common ancestor of the species we studied and then developed independently in different lineages of Ctenophores. So far, there is no evidence of additional expansion of the opsin family in ctenophore evolution. The involvement of ctenophore opsins in photoreception is discussed by analyzing their protein structures.

Due to their tissue structure similar to mammalian skin and their close evolutionary relationship with chordates, holothurians (Echinodermata: Holothuroidea) are particularly interesting for studies on wound healing. However, previous studies dealing with holothuroid wound healing have had limited approaches, being restricted to tissue repair or perivisceral immune response. In this study, we combined tissue, cellular and humoral parameters to study the wound healing process of Holothuria grisea. The immune responses of the perivisceral coelom were assessed by analyzing the number, proportion and viability of coelomocytes and the volume and protein concentration of the coelomic fluid. Additionally, the morphology of the healing tissue and number of coelomocytes in the connective tissue of different body wall layers were examined over 30 days. Our results showed that perivisceral reactions started 3 h after injury and decreased to baseline levels within 24 h. In contrast, tissue responses were delayed, beginning after 12 h and returning to baseline levels only after day 10. The number of coelomocytes in the connective tissue suggests a potential cooperation between these cells during wound healing: phagocytes and acidophilic spherulocytes act together in tissue clearance/homeostasis, whereas fibroblast-like and morula cells cooperate in tissue remodeling. Finally, our results indicate that the major phases observed in mammalian wound healing are also observed in H. grisea, despite occurring at a different timing, which might provide insights for future studies. Based on these data, we propose a model that explains the entire healing process in H. grisea.

Ecdysis-triggering hormone (ETH) is a neuropeptide hormone characterized by a conserved KxxKxxPRx amide structure widely identified in arthropods. While its involvement in the regulation of molting and reproduction in insects is well-established, its role in crustaceans has been overlooked. This study aimed to de-orphanise a receptor for ETH in the mud crab Scylla paramamosain and explore its potential impact on ovarian development. A 513-amino-acid G protein-coupled receptor for ETH (SpETHR) was identified in S. paramamosain, exhibiting a dose-dependent activation by SpETH with an EC₅₀ value of 75.18 nM. Tissue distribution analysis revealed SpETH was in the cerebral ganglion and thoracic ganglion, while SpETHR was specifically expressed in the ovary, hepatopancreas, and Y-organ of female crabs. In vitro experiments demonstrated that synthetic SpETH (at a concentration of 10⁻⁸ M) significantly increased the expression of SpVgR in the ovary and induced ecdysone biosynthesis in the Y-organ. In vivo experiments showed a significant upregulation of SpEcR in the ovary and Disembodied and Shadow in the Y-organ after 12 h of SpETH injection. Furthermore, a 16-day administration of SpETH significantly increased 20E titers in hemolymph, gonadosomatic index (GSI) and oocyte size of S. paramamosain. In conclusion, our findings suggest that SpETH may play stimulatory roles in ovarian development and ecdysone biosynthesis by the Y-organ.