Molecular Characterization of Branchial aquaporin 1aa and Effects of Seawater Acclimation,Emersion or Ammonia Exposure on Its mRNA Expression in the Gills,Gut, Kidney and Skin of the Freshwater Climbing Perch,Anabas testudineus

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH₃ transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.     

Phospholipid and Fatty Acid Composition of Phosphatidylcholine and Phosphatidylethanolamine in the Black Plaice <Emphasis Type="Italic">Pleuronectes obscura</Emphasis> during Thermoadaptation

The phospholipid (PL) and fatty acid (FA) composition of major membrane lipid constituents, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as the cholesterol/phospholipid (CL/PL) ratio were assayed in the muscles, gills and liver of the black plaice Pleuronectes (Liopsetta) obscura at different ambient temperatures (18, 9 and 0°C). PL and CL were shown to be actively involved in adaptation of the fish to changes in the seawater temperature. As temperature declines, the monounsaturated FA (MUFA) level increases while the polyunsaturated FA (PUFA) fraction in gills and liver PC and PE, on the contrary, decreases, resulting in diminished functional activity of the fish. However, in muscles this correlation is lacking. The PC and PE composition was shown to be organ- and ambient temperature-dependent. Major PC forms are saturated FA (SFA)/PUFA and MUFA/PUFA composed of a relatively small number of major molecular species. A temperature drop results in an increased SFA/PUFA level and decreased MUFA/PUFA and PUFA/PUFA levels in muscles and gills, and this may promote a drop in the viscosity of the outer lipid monolayer of membranes and in their functional activity. In contrast to PC, the PE composition in all organs tested is characterized by a decrease in the SFA/ PUFA level and an increase in MUFA/PUFA and PUFA/PUFA levels. Such changes promote the retention of functional activity of the inner lipid monolayer of membranes and are not synchronized with rearrangements in their outer monolayer. Due to intermolecular transfer of acyl radicals at a constancy of their composition, functional rearrangement of the lipid matrix appears to be achieved through changes in the membrane viscosity. Our data support the idea that different adaptation strategies in fish are driven by certain sets of PL molecular species.     

The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum)

Zanthoxylum armatum and Zanthoxylum bungeanum, known as ‘Chinese pepper’, are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.     

Long-Read Genome Sequencing Provides Molecular Insights into Scavenging and Societal Complexity in Spotted Hyena Crocuta crocuta

The spotted hyena (Crocuta crocuta) is a large and unique terrestrial carnivore. It is a particularly fascinating species due to its distinct phenotypic traits, especially its complex social structure and scavenging lifestyle, with associated high dietary exposure to microbial pathogens. However, the underlying molecular mechanisms related to these phenotypes remain elusive. Here, we sequenced and assembled a high-quality long-read genome of the spotted hyena, with a contig N50 length of ∼13.75 Mb. Based on comparative genomics, immunoglobulin family members (e.g., IGKV4-1) showed significant adaptive duplications in the spotted hyena and striped hyena. Furthermore, immune-related genes (e.g., CD8A, LAG3, and TLR3) experienced species-specific positive selection in the spotted hyena lineage. These results suggest that immune tolerance between the spotted hyena and closely related striped hyena has undergone adaptive divergence to cope with prolonged dietary exposure to microbial pathogens from scavenging. Furthermore, we provided the potential genetic insights underlying social complexity, hinting at social behavior and cognition. Specifically, the RECNE-associated genes (e.g., UGP2 and ACTR2) in the spotted hyena genome are involved in regulation of social communication. Taken together, our genomic analyses provide molecular insights into the scavenging lifestyle and societal complexity of spotted hyenas.     

Structural features of neural spines of the caudal vertebrae of protoceratopoids (Ornithischia: Neoceratopsia)

The structure of caudal neural spines of protoceratopoids displays adaptation for aquatic and terrestrial mode of life. The increasing height of caudal neural spines in the series Leptoceratops, Udanoceratops, Protoceratops, Bagaceratops is connected with the extent of adaptation for swimming and changes in inclination of neural spines are connected with the mechanical balance of the lever. Thus, the anterior caudal vertebrae (1cd–15cd) of Protoceratops and Bagaceratops show an anticliny, which promotes extension (rise) of a heavy tail in terrestrial conditions. In the middle part of the tail (16cd–23cd), with the greatest height of neural spines, a decrease in width and increase in thickness counteract transverse loads accompanying movements on land. At the same time, the supraspinal ligament prevents divergence of neural spines caused by curvature of the tail as it is raised above the ground.     

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae)

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish (Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 μg As/L). A control group was exposed to tap water (pH 8.0; 26 °C; 7.20 mg O₂/L). As exposure resulted in (1) an increase (p < 0.05) of glutathione (GSH) levels after exposure to 10 and 100 μg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations (p < 0.05), (3) no significant differences in terms of glutathione reductase, glutathione-S-transferase and catalase activities; (4) a significantly lower (p < 0.05) oxygen consumption after exposure to 100 μg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content (p > 0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.     

Effects of Cadmium and High Temperature on Some Parameters of Calcium Metabolism in the Killifish (Aphanius fasciatus)

This study aims to investigate the influence of high temperature on cadmium (Cd) toxicity in Aphanius fasciatus (Pisces: Cyprinodontidae). For this reason, Cd, mineral, and organic content in the vertebral column as well as the histological structure of gills and bone were compared in fishes exposed for 30 days to Cd (2 mg/L CdCl₂) and/or high temperature (26 °C). Cd exposure caused a negative correlation between Cd and Ca concentrations (r?=?0.98, p?<?0.05), as well as a significant decrease in inorganic components (p?<?0.05) and ash weight/dry weight ratio (p?<?0.05) in the vertebral column. These changes were accompanied by an increased frequency of histological alterations in gills and bone. Concomitant treatment with Cd and high temperature increases Cd accumulation and Ca depletion in the skeletal tissue and increases the frequency and the severity of histological alterations. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

Accelerated Evolutionary Rate of the Myoglobin Gene in Long-Diving Whales

Cetaceans, early in their evolutionary history, had developed many physiological adaptations to secondarily return to the sea. Among these adaptations, changes in molecules that transport oxygen and that ultimately support large periods of acute tissue hypoxia probably represent one big step toward the conquest of aquatic environments. Myoglobin contributes to intracellular oxygen storage and transcellular diffusion of oxygen in muscle, and plays an important role in supplying oxygen in hypoxic or ischemic conditions. Here we looked for evidence of adaptive molecular evolution of myoglobin in the cetacean lineage, relative to their terrestrial counterparts. We performed a comparative analysis to examine the variation of the parameter ω (d _N/d _S) and infer past period of adaptive evolution during the cetacean transition from the terrestrial to the aquatic environment. We also analyzed the changes in amino acid properties. At the nucleotide level, the results showed significant differences in selective pressure between cetacean and non-cetacean myoglobin (ω value three times higher in cetaceans when compared to terrestrial mammals), and also among cetacean lineages according to their diving capacities. Interestingly, both families with long duration diving cetaceans present two parallel substitutions (on sites 4 and 12). Regarding the amino acid properties, our analysis identified four significant physicochemical amino acid changes among residues in myoglobin protein under positive destabilizing selection.     

Combined use of leaf size and economics traits allows direct comparison of hydrophyte and terrestrial herbaceous adaptive strategies

Background and Aims

Hydrophytes generally exhibit highly acquisitive leaf economics. However, a range of growth forms is evident, from small, free-floating and rapidly growing Lemniden to large, broad-leaved Nymphaeiden, denoting variability in adaptive strategies. Traits used to classify adaptive strategies in terrestrial species, such as canopy height, are not applicable to hydrophytes. We hypothesize that hydrophyte leaf size traits and economics exhibit sufficient overlap with terrestrial species to allow a common classification of plant functional types, sensu Grime''s CSR theory.

Methods

Leaf morpho-functional traits were measured for 61 species from 47 water bodies in lowland continental, sub-alpine and alpine bioclimatic zones in southern Europe and compared against the full leaf economics spectrum and leaf size range of terrestrial herbs, and between hydrophyte growth forms.

Key Results

Hydrophytes differed in the ranges and mean values of traits compared with herbs, but principal components analysis (PCA) demonstrated that both groups shared axes of trait variability: PCA1 encompassed size variation (area and mass), and PCA2 ranged from relatively dense, carbon-rich leaves to nitrogen-rich leaves of high specific leaf area (SLA). Most growth forms exhibited trait syndromes directly equivalent to herbs classified as R adapted, although Nymphaeiden ranged between C and SR adaptation.

Conclusions

Our findings support the hypothesis that hydrophyte adaptive strategy variation reflects fundamental trade-offs in economics and size that govern all plants, and that hydrophyte adaptive strategies can be directly compared with terrestrial species by combining leaf economics and size traits.     

Metallothionein-like proteins in the blue crab Callinectes sapidus: Effect of water salinity and ions

The effect of water salinity and ions on metallothionein-like proteins (MTLP) concentration was evaluated in the blue crab Callinectes sapidus. MTLP concentration was measured in tissues (hepatopancreas and gills) of crabs acclimated to salinity 30 ppt and abruptly subjected to a hypo-osmotic shock (salinity 2 ppt). It was also measured in isolated gills (anterior and posterior) of crabs acclimated to salinity 30 ppt. Gills were perfused with and incubated in an isosmotic saline solution (ISS) or perfused with ISS and incubated in a hypo-osmotic saline solution (HSS). The effect of each single water ion on gill MTLP concentration was also analyzed in isolated and perfused gills through experiments of ion substitution in the incubation medium. In vivo, MTLP concentration was higher in hepatopancreas than in gills, being not affected by the hypo-osmotic shock. However, MTLP concentration in posterior and anterior gills significantly increased after 2 and 24 h of hypo-osmotic shock, respectively. In vitro, it was also increased when anterior and posterior gills were perfused with ISS and incubated in HSS. In isolated and perfused posterior gills, MTLP concentration was inversely correlated with the calcium concentration in the ISS used to incubate gills. Together, these findings indicate that an increased gill MTLP concentration in low salinity is an adaptive response of the blue crab C. sapidus to the hypo-osmotic stress. This response is mediated, at least in part, by the calcium concentration in the gill bath medium. The data also suggest that the trigger for this increase is purely branchial and not systemic.     

Gill Phospholipids of Mitochondria in Euryhaline Crustaceans as Related to Changes in Environmental Salinity

The distribution of different phospholipids and their variation in fatty acids composition were studied in mitochondrial fractions isolated from anterior and posterior gills of the two euryhaline crabs, Enocheir sinensis and Carcinus maenas, as a function of the environmental salinity. No matter what the salinity, the three more posterior located gills of E. sinensis were shown to contain more unsaturated phospholipids (PE, DPG) and more eicosapentaanoic acids (20:5ω 3) than the three more anterior ones. This was particularly significant when crabs were acclimatized to fresh water. The lipid content of the anterior and posterior gills of the seashore crab C. maenas, on the contrary, showed no significant differences. These results are discussed by taking into consideration the different osmo- and ion-regulation capabilities of the two euryhaline crabs studied and it is proposed that a possible viscotropic regulation might check the activity of membrane-bound enzymes among which the (Na⁺ + K⁺)-ATPase related to the Na⁺-active transport processes involved in maintaining Na⁺ balance.     

The morphology and vasculature of the respiratory organs of terrestrial hermit crabs (Coenobita and Birgus): gills,branchiostegal lungs and abdominal lungs

The morphology and vasculature of the respiratory organs of the terrestrial coenobitids were studied using light microscopy, TEM, SEM and corrosion casting. The gills of Coenobita and Birgus are modified for air-breathing but are reduced in number and size and have a comparatively small surface area. The branchiostegal lungs of Coenobita (which live in gastropod shells) are very small but are well vascularized and have a thin blood/gas barrier. Coenobita has developed a third respiratory organ, the abdominal lung, that is formed from highly vascularized patches of very thin and intensely-folded dorsal integument. Oxygenated blood from this respiratory surface is returned to the pericardial sinus via the gills (in parallel to the branchiostegal circulation). Birgus, which does not inhabit a gastropod shell, has developed a highly complex branchiostegal lung that is expanded laterally and evaginated to increase surface area. The blood/gas diffusion distance is short and oxygenated blood is returned directly to the pericardium via pulmonary veins. We conclude that the presence of a protective mollusc shell in the terrestrial hermit crabs has favoured the evolution of an abdominal lung and in its absence a branchiostegal lung has been developed.     

Damage to the gills and integument of Litoria fallax larvae (Amphibia: Anura) associated with ionoregulatory disturbance at low pH

In fish, exposure to waters of low pH causes significant damage to the gill resulting in fatal iono- and osmoregulatory disturbance. In amphibians, exposure to acid waters also disrupts ionic homeostasis, however the extent and nature of injuries to amphibian larvae from acid exposure are poorly understood. Changes in gross morphology and ultrastructure of the gills and integument were examined, together with measures of Na⁺ efflux/uptake, in larval Litoria fallax (Amphibia: Anura) following acute acid exposure. Examination of tissues revealed significant changes in morphology and ultrastructure of both gills and the integument following acutely lethal exposure to low pH water. Changes to the gills of acid-exposed L. fallax larvae included lifting of the branchial epithelium and opening of tight junctions between pavement cells (with a consequent reduction in tight junction length). Damage to epithelial cell–cell tight junctions was also apparent at the integument along with widespread oncosis and localised epithelial necrosis. Mucous secretory activity at the gills and body surface was largely unaffected by acid exposure, with little or no difference in density, cross-sectional area and number of epithelial mucous secretory vesicles in acid-exposed and control larvae. Changes in morphology and ultrastructure at low pH were accompanied by significant Na⁺ loss (up to 50% of the total body Na⁺ content) attributable in large part to increased paracellular ionic efflux across the gills as well as increased transcellular and paracellular efflux of ions across the integument.     

Was the Early Eocene proboscidean Numidotherium koholense semi-aquatic or terrestrial? Evidence from stable isotopes and bone histology

The Early Eocene deposits of El Kohol, Algeria, have yielded numerous remains of Numidotherium koholense, one of the most primitive and oldest known proboscideans in Africa. The Upper Eocene proboscideans of the Fayum locality (Egypt), Barytherium sp. and Moeritherium sp., were recently interpreted as aquatic or semi-aquatic, according to the stable isotopic compositions (δ¹³C and δ¹⁸O) of their tooth enamel. These data led us to reinvestigate the adaptations of N. koholense. Stable isotopic analysis and observations of histological sections of its long bones reveal that it was essentially terrestrial. According to its position within the phylogenetic tree of Eocene proboscideans, the adaptation to semi-aquatic life appears to have evolved independently in different lineages of Middle and Upper Eocene proboscideans during their adaptive radiation in Africa. Moreover, these new results reopen the debate about the hypothesis that Eocene to Recent proboscideans are derived from semi-aquatic ancestors.