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Gut microbiota studies often rely on a single sample taken per individual, representing a snapshot in time. However, we know that gut microbiota composition in many animals exhibits intra-individual variation over the course of days to months. Such temporal variations can be a confounding factor in studies seeking to compare the gut microbiota of different wild populations, or to assess the impact of medical/veterinary interventions. To date, little is known about the variability of the koala ( Phascolarctos cinereus ) gut microbiota through time. Here, we characterise the gut microbiota from faecal samples collected at eight timepoints over a month for a captive population of South Australian koalas ( n individuals = 7), and monthly over 7 months for a wild population of New South Wales koalas ( n individuals = 5). Using 16S rRNA gene sequencing, we found that microbial diversity was stable over the course of days to months. Each koala had a distinct faecal microbiota composition which in the captive koalas was stable across days. The wild koalas showed more variation across months, although each individual still maintained a distinct microbial composition. Per koala, an average of 57 (±16) amplicon sequence variants (ASVs) were detected across all time points; these ASVs accounted for an average of 97% (±1.9%) of the faecal microbial community per koala. The koala faecal microbiota exhibits stability over the course of days to months. Such knowledge will be useful for future studies comparing koala populations and developing microbiota interventions for this regionally endangered marsupial., Competing Interests: Kellie Leigh is employed by Science for Wildlife Ltd., (© 2023 Eisenhofer et al.)

COVID-19 has devastated global communities and economies. The pandemic has exposed socioeconomic disparities and weaknesses in health systems worldwide. Long-term health effects and economic recovery are major concerns. Ecosystem restoration-ie, the repair of ecosystems that have been degraded-relates directly to tackling the health and socioeconomic burdens of COVID-19, because stable and resilient ecosystems are fundamental determinants of health and socioeconomic stability. Here, we use COVID-19 as a case study, showing how ecosystem restoration can reduce the risk of infection and adverse sequelae and have an integral role in humanity's recovery from COVID-19. The next decade will be crucial for humanity's recovery from COVID-19 and for ecosystem repair. Indeed, in the absence of effective, large-scale restoration, 95% of the Earth's land could be degraded by 2050. The UN Decade on Ecosystem Restoration (2021-30) declaration reflects the growing urgency and scale at which we should repair ecosystems. Importantly, ecosystem restoration could also help to combat the health and socioeconomic issues that are associated with COVID-19, yet it is poorly integrated into current responses to the disease. Ecosystem restoration can be a core public health intervention and assist in COVID-19 recovery if it is closely integrated with socioeconomic, health, and environmental policies., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Although the restorative benefits of nature are widely acknowledged, there is a limited understanding of the attributes of natural environments that are fundamental to restorative experiences. Faced with growing human populations and a greater awareness of the wellbeing benefits natural environments provide, park agencies and planners are increasingly challenged with balancing human and ecological outcomes in natural areas. This study examines the physical and experiential qualities of natural environments people referred to when describing their connection to their most valued natural environments in an online questionnaire. Recruited primarily via a public radio program, respondents were asked to identify their favorite places and explain what they loved about those places. Favorite places are considered exemplars of restorative environments and were classified based on an existing park typology. Reasons people liked particular sites were classified into three domains: setting, activity, or benefit. Content analysis was used to identify the attributes most commonly associated with favorite places. These attributes were then related to the four components of restorative environments according to Attention Restoration Theory. In contrast to previous research, we found that "fascination" was the most important component of favorite places. Possible reasons for this contrast, namely, respondents' median age, and the likelihood of a high degree of ecological literacy amongst the study population are discussed. South Australians' favorite environments comprise primarily hilly, wooded nature parks, and botanical gardens, in stark contrast to the vast arid areas that dominate the state. Micro-variables such as birds, plants, wildlife, native species, and biodiversity appear particularly important elements used to explain people's love of these sites. We discuss the implications of these findings and their potential value as an anchor for marketing campaigns seeking to encourage contact with nature, as well as education programs designed to improve people's understanding of important but intangible concepts such as biodiversity. The findings have clear, practical implications for park managers given the modifiable nature of many of the attributes identified as being most important to our respondents, and we believe attention to such elements has the potential to simultaneously enhance people's nature experiences, optimize restorative outcomes, and improve environmental stewardship.

Knowledge and understanding about how the Earth functions and supports life create the foundation for ecological literacy. Industrialisation, urbanisation and population growth have resulted in changed relationships between many human communities and the natural world. A potential consequence is a compromised capability to make well-informed decisions about how to live sustainably. To gain a measure of ecological literacy within the South Australian community, we collaborated with senior scientists and educators to develop and apply an instrument with the capacity to determine indicative levels of ecological knowledge and understanding. A formal, variable credit, multiple-choice assessment instrument was distributed online to groups and individuals within diverse community sectors and industries. Quantitative analyses of scores indicated that levels of ecological knowledge and understanding within a self-selected sample of over one thousand individuals ranged from very low to extremely high, with the majority of respondents achieving moderate to high scores. This instrument has a demonstrated capacity to determine indicative levels of ecological literacy within and between individuals and groups. It is able to capture mastery of ecological knowledge and understanding achieved through both formal and informal pathways. Using the results, we have been able to establish a range of standards and an aspirational target score for the South Australian community. The value of this work is in its potential to deliver insights into relationships between humans and the rest of the natural world, and into characteristics of eco-literate individuals and communities, that might not otherwise emerge.

Surfactant lipids and proteins form a surface active film at the air-liquid interface of internal gas exchange organs, including swim bladders and lungs. The system is uniquely positioned to meet both the physical challenges associated with a dynamically changing internal air-liquid interface, and the environmental challenges associated with the foreign pathogens and particles to which the internal surface is exposed. Lungs range from simple, transparent, bag-like units to complex, multilobed, compartmentalized structures. Despite this anatomical variability, the surfactant system is remarkably conserved. Here, we discuss the evolutionary origin of the surfactant system, which likely predates lungs. We describe the evolution of surfactant structure and function in invertebrates and vertebrates. We focus on changes in lipid and protein composition and surfactant function from its antiadhesive and innate immune to its alveolar stability and structural integrity functions. We discuss the biochemical, hormonal, autonomic, and mechanical factors that regulate normal surfactant secretion in mature animals. We present an analysis of the ontogeny of surfactant development among the vertebrates and the contribution of different regulatory mechanisms that control this development. We also discuss environmental (oxygen), hormonal and biochemical (glucocorticoids and glucose) and pollutant (maternal smoking, alcohol, and common "recreational" drugs) effects that impact surfactant development. On the adult surfactant system, we focus on environmental variables including temperature, pressure, and hypoxia that have shaped its evolution and we discuss the resultant biochemical, biophysical, and cellular adaptations. Finally, we discuss the effect of major modern gaseous and particulate pollutants on the lung and surfactant system., (Copyright © 2015 John Wiley & Sons, Inc.)

The koala (Phascolarctos cinereus) occurs in the eucalypt forests of eastern and southern Australia and is currently threatened by habitat fragmentation, climate change, sexually transmitted diseases, and low genetic variability throughout most of its range. Using data collected during the Great Koala Count (a 1-day citizen science project in the state of South Australia), we developed generalized linear mixed-effects models to predict habitat suitability across South Australia accounting for potential errors associated with the dataset. We derived spatial environmental predictors for vegetation (based on dominant species of Eucalyptus or other vegetation), topographic water features, rain, elevation, and temperature range. We also included predictors accounting for human disturbance based on transport infrastructure (sealed and unsealed roads). We generated random pseudo-absences to account for the high prevalence bias typical of citizen-collected data. We accounted for biased sampling effort along sealed and unsealed roads by including an offset for distance to transport infrastructures. The model with the highest statistical support (wAIC c ∼ 1) included all variables except rain, which was highly correlated with elevation. The same model also explained the highest deviance (61.6%), resulted in high R (2)(m) (76.4) and R (2)(c) (81.0), and had a good performance according to Cohen's κ (0.46). Cross-validation error was low (∼ 0.1). Temperature range, elevation, and rain were the best predictors of koala occurrence. Our models predict high habitat suitability in Kangaroo Island, along the Mount Lofty Ranges, and at the tips of the Eyre, Yorke and Fleurieu Peninsulas. In the highest-density region (5576 km(2)) of the Adelaide-Mount Lofty Ranges, a density-suitability relationship predicts a population of 113,704 (95% confidence interval: 27,685-199,723; average density = 5.0-35.8 km(-2)). We demonstrate the power of citizen science data for predicting species' distributions provided that the statistical approaches applied account for some uncertainties and potential biases. A future improvement to citizen science surveys to provide better data on search effort is that smartphone apps could be activated at the start of the search. The results of our models provide preliminary ranges of habitat suitability and population size for a species for which previous data have been difficult or impossible to gather otherwise.

Urban environments are unique, rapidly changing habitats in which almost half of the world's human population resides. The effects of urbanisation, such as habitat (vegetation) removal, pollution and modification of natural areas, commonly cause biodiversity loss. Long-term ecological monitoring of urban environments is vital to determine the composition and long-term trends of faunal communities. This paper provides a detailed view of long-term changes in avifaunal assemblages of the Adelaide City parklands and discusses the anthropogenic and environmental factors that contributed to the changes between 1976 and 2007. The Adelaide City parklands (ACP) comprise 760 ha of land surrounding Adelaide's central business district. Naturalist Robert Whatmough completed a 32-year survey of the ACP to determine the structure of the urban bird community residing there. Annual species richness and the abundance of birds in March and September months were analysed. Linear regression analysis was applied to species richness and abundance data of each assemblage. Resident parkland birds demonstrated significant declines in abundance. Native and introduced species also exhibited long-term declines in species richness and abundance throughout the 32-year period. Cycles of varying time periods indicated fluctuations in avian biodiversity demonstrating the need for future monitoring and statistical analyses on bird communities in the Adelaide City parklands.

Pulmonary surfactant fulfils diverse functions at the lung air-liquid interface of all air-breathing vertebrates. Neurohormonal regulation of surfactant synthesis and secretion is highly conserved among non-mammalian amniotes. Although the pattern of surfactant lipid maturation is similar among species, the onset and completion differ dramatically. These differences are apparently not determined by phylogeny, but may relate to the timing of development of relative hypoxia as an embryo develops, which is related to birthing strategy. We have proposed that hypoxia is an evolutionary drive for differential surfactant development among species. In mammalian and non-mammalian models, hypoxia induces fetal growth restriction. Depending on the timing of the insult, this may be associated with an acceleration or deceleration of surfactant development. The hypoxic effect may be mediated via hormonal and growth factors, such as glucocorticoids and VEGF. However, the multifactorial nature of mammalian growth restriction models complicates the mechanistic interpretations. Hence, less complex oviparous animal models are required, in which hypoxia can be isolated from maternal influences., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recent findings are reported about certain aspects of the structure and function of the mammalian and avian lungs that include (a) the architecture of the air capillaries (ACs) and the blood capillaries (BCs); (b) the pulmonary blood capillary circulatory dynamics; (c) the adaptive molecular, cellular, biochemical, compositional, and developmental characteristics of the surfactant system; (d) the mechanisms of the translocation of fine and ultrafine particles across the airway epithelial barrier; and (e) the particle-cell interactions in the pulmonary airways. In the lung of the Muscovy duck Cairina moschata, at least, the ACs are rotund structures that are interconnected by narrow cylindrical sections, while the BCs comprise segments that are almost as long as they are wide. In contrast to the mammalian pulmonary BCs, which are highly compliant, those of birds practically behave like rigid tubes. Diving pressure has been a very powerful directional selection force that has influenced phenotypic changes in surfactant composition and function in lungs of marine mammals. After nanosized particulates are deposited on the respiratory tract of healthy human subjects, some reach organs such as the brain with potentially serious health implications. Finally, in the mammalian lung, dendritic cells of the pulmonary airways are powerful agents in engulfing deposited particles, and in birds, macrophages and erythrocytes are ardent phagocytizing cellular agents. The morphology of the lung that allows it to perform different functions-including gas exchange, ventilation of the lung by being compliant, defense, and secretion of important pharmacological factors-is reflected in its "compromise design."

Elasmosaurs were extremely long-necked, aquatic reptiles that used four flippers for locomotion. Their distinctive long neck distinguishes them from all other Mesozoic forms, yet the potential uses and constraints of this structure are poorly understood, particularly with regard to feeding. Several associated series of elasmosaurian cervical vertebrae were used to measure ranges of potential flexion. Two-dimensional models, based on a complete specimen of the Late Cretaceous elasmosaur Aphrosaurus furlongi, were created to measure mobility in both vertical and horizontal planes. Accuracy of the models was assessed through comparative analyses with currently extant vertebrate analogues (e.g. snake, turtle, seal). Results suggest that the elasmosaurian neck was capable of a 75-177 degrees ventral, 87-155 degrees dorsal, and 94-176 degrees lateral range of movement depending upon the thickness of cartilage reconstructed between each vertebra. Neck postures such as a 'swan-like' S-shape are shown to be implausible because they require >360 degrees vertical flexion. However, maintenance of a straight neck while swimming, together with considerable lateral and/or ventral movement during prey capture and feeding are feasible.

(Orgeig and Daniels) This surfactant symposium reflects the integrative and multidisciplinary aims of the 1st ICRB, by encompassing in vitro and in vivo research, studies of vertebrates and invertebrates, and research across multiple disciplines. We explore the physical and structural challenges that face gas exchange surfaces in vertebrates and insects, by focusing on the role of the surfactant system. Pulmonary surfactant is a complex mixture of lipids and proteins that lines the air-liquid interface of the lungs of all air-breathing vertebrates, where it functions to vary surface tension with changing lung volume. We begin with a discussion of the extraordinary conservation of the blood-gas barrier among vertebrate respiratory organs, which has evolved to be extremely thin, thereby maximizing gas exchange, but simultaneously strong enough to withstand significant distension forces. The principal components of pulmonary surfactant are highly conserved, with a mixed phospholipid and neutral lipid interfacial film that is established, maintained and dynamically regulated by surfactant proteins (SP). A wide variation in the concentrations of individual components exists, however, and highlights lipidomic as well as proteomic adaptations to different physiological needs. As SP-B deficiency in mammals is lethal, oxidative stress to SP-B is detrimental to the biophysical function of pulmonary surfactant and SP-B is evolutionarily conserved across the vertebrates. It is likely that SP-B was essential for the evolutionary origin of pulmonary surfactant. We discuss three specific issues of the surfactant system to illustrate the diversity of function in animal respiratory structures. (1) Temperature: In vitro analyses of the behavior of different model surfactant films under dynamic conditions of surface tension and temperature suggest that, contrary to previous beliefs, the alveolar film may not have to be substantially enriched in the disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), but that similar properties of rate of film formation can be achieved with more fluid films. Using an in vivo model of temperature change, a mammal that enters torpor, we show that film structure and function varies between surfactants isolated from torpid and active animals. (2) Spheres versus tubes: Surfactant is essential for lung stabilization in vertebrates, but its function is not restricted to the spherical alveolus. Instead, surfactant is also important in narrow tubular respiratory structures such as the terminal airways of mammals and the air capillaries of birds. (3). Insect tracheoles: We investigate the structure and function of the insect tracheal system and ask whether pulmonary surfactant also has a role in stabilizing these minute tubules. Our theoretical analysis suggests that a surfactant system may be required, in order to cope with surface tension during processes, such as molting, when the tracheae collapse and fill with water. Hence, despite observations by Wigglesworth in the 1930s of fluid-filled tracheoles, the challenge persists into the 21st century to determine whether this fluid is associated with a pulmonary-type surfactant system. Finally, we summarize the current status of the field and provide ideas for future research.

Maximum-likelihood models of codon and amino acid substitution were used to analyze the lung-specific surfactant protein C (SP-C) from terrestrial, semi-aquatic, and diving mammals to identify lineages and amino acid sites under positive selection. Site models used the nonsynonymous/synonymous rate ratio (omega) as an indicator of selection pressure. Mechanistic models used physicochemical distances between amino acid substitutions to specify nonsynonymous substitution rates. Site models strongly identified positive selection at different sites in the polar N-terminal extramembrane domain of SP-C in the three diving lineages: site 2 in the cetaceans (whales and dolphins), sites 7, 9, and 10 in the pinnipeds (seals and sea lions), and sites 2, 9, and 10 in the sirenians (dugongs and manatees). The only semi-aquatic contrast to indicate positive selection at site 10 was that including the polar bear, which had the largest body mass of the semi-aquatic species. Analysis of the biophysical properties that were influential in determining the amino acid substitutions showed that isoelectric point, chemical composition of the side chain, polarity, and hydrophobicity were the crucial determinants. Amino acid substitutions at these sites may lead to stronger binding of the N-terminal domain to the surfactant phospholipid film and to increased adsorption of the protein to the air-liquid interface. Both properties are advantageous for the repeated collapse and reinflation of the lung upon diving and resurfacing and may reflect adaptations to the high hydrostatic pressures experienced during diving.

The pulmonary surfactant system of heterothermic mammals must be capable of dealing with the effect of low body temperatures on the physical state of the lipid components. We have shown previously that there is a modest increase in surfactant cholesterol during periods of torpor, however these changes do not fully explain the capacity of surfactant to function under the wide range of physical conditions imposed by torpor. Here we examine indirectly the role of surfactant protein C (SP-C) in adapting to variable body temperatures by testing for the presence of positive (adaptive) selection during evolutionary transitions between heterothermy and homeothermy. We sequenced SP-C from genomic DNA of 32 mammalian species from groups of closely related heterothermic and homeothermic species (contrasts). We used phylogenetic analysis by maximum likelihood estimates of rates of non-synonymous to synonymous substitutions and fully Bayesian inference of these sequences to determine whether the mode of body temperature regulation exerts a selection pressure driving the molecular adaptation of SP-C. The protein sequence of SP-C is highly conserved with synonymous or highly conservative amino acid substitutions being predominant. The evolution of SP-C among mammals is characterised by high codon usage bias and high rates of transition/transversion. The only contrast to show evidence of positive selection was that of the bears (Ursus americanus and U. maritimus). The significance of this result is unclear. We show that SP-C is under strong evolutionary constraints, driven by purifying selection, presumably to maintain protein function despite variation in the mode of body temperature regulation.

Rational treatment of lymphoedema may be improved in the future with a better understanding of the physiological processes involved in the regeneration of new lymphatic vessels (lymphangiogenesis). Many lizard species undergo tail autotomy as a predator escape response and subsequently regenerate nonlymphoedematous tails. Such species may offer novel models for examining lymphangiogenesis. In this lymphoscintigraphic evaluation, three radioactive tracers were employed, (99m)Tc-antimony trisulphide colloid (approximately 10 nm diameter), (99m)Tc-tin fluoride colloid (approximately 2,000 nm; (99m)Tc-TFC), and (99m)Tc-diethylenetriaminepentaacetic acid (soluble; (99m)Tc-DTPA), to examine lymphatic function in regenerating tails of the Australian marbled gecko, Christinus marmoratus. Rate of local clearance and velocity of migration were determined in geckos with original tails and at 6, 9, 12, and >24 weeks after autotomy. In original-tailed geckos, the smaller radiocolloid was cleared to a greater extent and had a faster lymph velocity than in geckos with regenerated tails. The same parameters measured for larger particles were greater in early regeneration than later. (99m)Tc-TFC did not migrate from the injection site in fully regenerated and original gecko tails, which indicates that larger particles are increasingly impeded as tail regeneration progresses. Soluble (99m)Tc-DTPA diffused from the injection site extremely rapidly via venous capillaries in all tails, confirming that the slower clearance of the colloids is solely via the lymphatics. Differences in clearance and lymph velocity between differently sized colloids throughout tail regeneration may be influenced by changes in surrounding tissue structure density and the lymphatic vessel porosity., (c 2006 Wiley-Liss, Inc.)

Pulmonary surfactant lines the alveolar air-water interface, varying surface tension with lung volume to increase compliance and prevent adhesion of respiratory surfaces. We examined whether the surfactant system of diving mammals exhibits adaptations for more efficient lung function during diving, to complement other respiratory adaptations. Here we review adaptations at the molecular, compositional, functional and cellular levels and during development for animals beginning life on land and progressing to an aquatic environment. Molecular adaptations to diving were examined in surfactant protein C (SP-C) from terrestrial, semi-aquatic and diving mammals using phylogenetic analyses. Diving species exhibited sites under positive selection in the polar N-terminal domain. These amino acid substitutions may lead to stronger binding of SP-C to the phospholipid film and increased adsorption to the air-liquid interface. The concentration of shorter chain phospholipid molecular species was greater and SP-B levels were lower in diving than terrestrial mammals. This may lead to a greater fluidity and explain the relatively poor surface activity of diving mammal surfactant. There were no consistent differences in cholesterol between diving and terrestrial mammals. Surfactant from newborn California sea lions was similar to that of terrestrial mammals. Secretory activity of alveolar type II epithelial cells of sea lions demonstrated an insensitivity to pressure relative to sheep cells. The poor surface activity of diving mammal surfactant is consistent with the hypothesis that it has an anti-adhesive function that develops after the first entry into the water, with a surfactant film that is better suited to repeated collapse and respreading.

The genotoxic potential of sediment pore water collected from coastal areas in the Gulf of Mexico has not been reported frequently in the literature. This report summarizes a study of the microbial mutagenicity of 31 pore water samples obtained from sediment affected by non-point source runoff and compares the results with those for more traditional chemical and biological indicators of sediment quality. Genotoxicity was determined pre- and post-enzyme activation using a proprietary short-term microbial assay for pore water centrifuged from sediment collected adjacent to a Florida coastal golf complex and from an urbanized bayou-estuary. Sediment and the associated pore water also were analyzed for acute toxicity to Hyallela azteca, Palaemonetes pugio, or Americamysis bahia and for benthic macroinvertebrate diversity (sediment only). Genotoxicity (direct and enzyme-activated) was detected in 4 of 17 (golf complex) and in 10 of 14 (urbanized bayou) pore water samples. The lowest toxic pore water concentrations were between 1.8% and 44.4% (direct) and between 2.6% and 25% (enzyme-activated). The results of the genotoxic assay paralleled those based on exceedance of proposed sediment quality guidelines, pore water acute toxicity and Shannon-Wiener diversity index values for 81%, 58%, and 65% of the comparisons, respectively., (Copyright 2006 Wiley Periodicals, Inc.)

Maintaining a functional pulmonary surfactant system at depth is critical for diving mammals to ensure that inspiration is possible upon re-emergence. The lipid and protein composition of lavage extracts from three pinniped species (California sea lion, Northern elephant seal and Ringed seal) were compared to several terrestrial species. Lavage samples were purified using a NaBr discontinuous gradient. Concentrations of phospholipid classes and molecular species were measured using electrospray ionisation mass spectrometry, cholesterol was measured using high-performance liquid chromatography, surfactant protein A (SP-A) and SP-B were measured using enzyme-linked immunosorbent assays. There were small differences in phospholipid classes, with a lower level of anionic surfactant phospholipids, PG and PI, between diving and terrestrial mammals. There were no differences in PL saturation or SP-A levels between species. PC16:0/14:0, PC16:0/16:1, PC16:0/16:0, long chain PI species and the total concentrations of alkyl-acyl species of PC and PG as a ratio of diacyl species were increased in diving mammals, whereas concentrations of PC16:0/18:1, PG16:0/16:0 and PG16:0/18:1 were decreased. Cholesterol levels were very variable between species and SP-B was very low in diving mammals. These differences may explain the very poor surface activity of pinniped surfactant that we have previously described [Miller, N.J., Daniels, C.B., Schürch, S., Schoel, W.M., Orgeig, S., 2005. The surface activity of pulmonary surfactant from diving mammals. Respir. Physiol. Neurobiol. 150 (2006) 220-232], supporting the hypothesis that pinniped surfactant has primarily an anti-adhesive function to meet the challenges of regularly collapsing lungs.

Pinnipeds (seals and sea lions) have developed a specialised respiratory system to cope with living in a marine environment. They have a highly reinforced lung that can completely collapse and reinflate during diving without any apparent side effects. These animals may also have a specialised surfactant system to augment the morphological adaptations. The surface activity of surfactant from four species of pinniped (California sea lion, Northern elephant seal, Northern fur seal and Ringed seal) was measured using a captive bubble surfactometer (CBS), and compared to two terrestrial species (sheep and cow). The surfactant of Northern elephant seal, Northern fur seal and Ringed seal was unable to reduce surface tension (gamma) to normal levels after 5 min adsorption (61.2, 36.7, and 46.2 +/- 1.7 mN/m, respectively), but California sea lion was able to reach the levels of the cow and sheep (23.4 mN/m for California sea lion, 21.6 +/- 0.3 and 23.0 +/- 1.5 mN/m for cow and sheep, respectively). All pinnipeds were also unable to obtain the very low gamma(min) achieved by cow (1.4 +/- 0.1 mN/m) and sheep (1.5 +/- 0.4 mN/m). These results suggest that reducing surface tension to very low values is not the primary function of surfactant in pinnipeds as it is in terrestrial mammals, but that an anti-adhesive surfactant is more important to enable the lungs to reopen following collapse during deep diving.

Pulmonary surfactant, a complex mixture of lipids and proteins, lowers the surface tension in terminal air spaces and is crucial for lung function. Within an animal species, surfactant composition can be influenced by development, disease, respiratory rate, and/or body temperature. Here, we analyzed the composition of surfactant in three heterothermic mammals (dunnart, bat, squirrel), displaying different torpor patterns, to determine: 1) whether increases in surfactant cholesterol (Chol) and phospholipid (PL) saturation occur during long-term torpor in squirrels, as in bats and dunnarts; 2) whether surfactant proteins change during torpor; and 3) whether PL molecular species (molsp) composition is altered. In addition, we analyzed the molsp composition of a further nine mammals (including placental/marsupial and hetero-/homeothermic contrasts) to determine whether phylogeny or thermal behavior determines molsp composition in mammals. We discovered that like bats and dunnarts, surfactant Chol increases during torpor in squirrels. However, changes in PL saturation during torpor may not be universal. Torpor was accompanied by a decrease in surfactant protein A in dunnarts and squirrels, but not in bats, whereas surfactant protein B did not change in any species. Phosphatidylcholine (PC)16:0/16:0 is highly variable between mammals and is not the major PL in the wombat, dunnart, shrew, or Tasmanian devil. An inverse relationship exists between PC16:0/16:0 and two of the major fluidizing components, PC16:0/16:1 and PC16:0/14:0. The PL molsp profile of an animal species is not determined by phylogeny or thermal behavior. We conclude that there is no single PL molsp composition that functions optimally in all mammals; rather, surfactant from each animal is unique and tailored to the biology of that animal.

Pulmonary surfactant has previously been shown to change during development, both in composition and function. Adult pinnipeds, unlike adult terrestrial mammals, have an altered lung physiology to cope with the high pressures associated with deep diving. Here, we investigated how surfactant composition and function develop in California sea lions (Zalophus californianus). Phosphatidylinositol was the major anionic phospholipid in the newborn, whereas phosphatidylglycerol was increased in the adult. This increase in phosphatidylglycerol occurred at the expense of phosphatidylinositol and phosphatidylserine. There was a shift from long chain and polyunsaturated phospholipid molecular species in the newborn to shorter chain and mono- and disaturated molecular species in the adult. Cholesterol and SP-B concentrations were also higher in the adult. Adult surfactant could reach a lower equilibrium surface tension, but newborn surfactant could reach a lower minimum surface tension. The composition and function of surfactant from newborn California sea lions suggest that this age group is similar to terrestrial newborn mammals, whereas the adult has a "diving mammal" surfactant that can aid the lung during deep dives. The onset of diving is probably a trigger for surfactant development in these animals.

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish (the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs (A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

The surfactant system, a complex mixture of lipids and proteins, controls surface tension in the lung and is crucial for the first breath at birth, and thereafter. Heterokairy is defined as plasticity of a developmental process within an individual. Here, we provide experimental evidence for the concept of heterokairy, as hypoxia induces a change in the onset and rate of development of surfactant, probably via endogenous glucocorticoids, to produce individuals capable of surviving early hatching. Chicken eggs were incubated under normoxic (21% O(2)) conditions throughout or under hypoxic (17% O(2)) conditions from day 10 of incubation. Embryos were sampled at days 16, 18, and 20 and also 24 h after hatching. In a second experiment, dexamethasone (Dex), tri-iodothyronine (T(3)), or a combination (Dex + T(3)) was administered 24 and 48 h before each time point. Both hypoxia and Dex accelerated maturation of the surfactant lipids by increasing total phospholipid (PL), disaturated phospholipid (DSP), and cholesterol (Chol) in lavage at days 16 and 18. Maturation of surfactant lipid composition was accelerated, with day 16 %DSP/PL, Chol/DSP, and Chol/PL resembling the ratios of day 20 control animals. The effect of Dex + T(3) was similar to that of Dex alone. Hypoxia increased plasma corticosterone levels at day 16, while plasma T(3) levels were not affected. Hence, exposure to hypoxia during critical developmental windows accelerates surfactant maturation, probably by increasing corticosterone production. This internal modulation of the developmental response to an external stimulus is a demonstration of physiological heterokairy.

Marine mammals have a spectacular suite of respiratory adaptations to deal with the extreme pressures associated with deep diving. In particular, maintaining a functional pulmonary surfactant system at depth is critical for marine mammals to ensure that inspiration is possible upon re-emergence. Pulmonary surfactant is secreted from alveolar type II (ATII) cells and is crucial for normal lung function. It is not known whether ATII cells have the ability to continue to secrete pulmonary surfactant under pressure, or how secretion is maintained and controlled. We show here that surfactant secretion in California sea lions (Zalophus californianus) was increased after high pressures (25 and 50 atm) of short duration (30 min), but was unaffected by high pressures of long duration (2 h). This is in contrast to a similar sized terrestrial mammal (sheep), where surfactant secretion was increased after high pressures of both long and short duration. Z. californianus and terrestrial mammals also show similar responses to stimulatory hormones and autonomic neurotransmitters. It therefore seems that an increase in the quantity of surfactant in seal lungs after diving is most likely caused by mechanostimulation induced by pressure and volume changes, and that seals are adapted to maintain constant levels of surfactant under long periods of high pressure.

Fat-tailed dunnarts, Sminthopsis crassicaudata, survive dramatic changes in body temperature during torpor without experiencing surfactant dysfunction. Adrenergic factors regulate surfactant secretion through beta(2)-adrenergic receptors on alveolar type II cells. Temperature has no effect on the secretory response of dunnart type II cells to adrenergic stimulation. We hypothesise that during torpor, dunnart type II cells up-regulate the number of adrenergic receptors present on type II cells to enable stimulation at lower concentrations of agonist. Here, we isolated type II cells from warm-active (35 degrees C) and torpid (15 degrees C) dunnarts and examined the effects of an in vitro temperature change on the number and activity of adrenergic receptors. Torpor did not affect the beta-adrenergic receptor number. However, we observed a significant decrease in adrenergic receptor number when cells from warm-active animals were incubated at 15 degrees C and when cells from torpid animals were incubated at 37 degrees C. cAMP production was significantly higher in type II cells from torpid dunnarts than warm-active dunnarts and this may contribute, in part, to the temperature insensitivity we have previously observed in the adrenergic regulation of surfactant secretion.

Homeothermic mammals experience pulmonary surfactant dysfunction with relatively small fluctuations in body temperature. However, ground squirrels survive dramatic changes in body temperature during hibernation, when body temperature drops from 37 degrees C to 0-5 degrees C during prolonged torpor bouts. Using type II cells isolated from both warm-active and torpid squirrels, we determined the effect of assay temperature, autonomic agonists and torpor on surfactant secretion. Basal secretion was significantly higher in type II cells isolated from torpid squirrels compared with warm-active squirrels when assayed at the body temperature of the animal from which they were isolated (4 degrees C and 37 degrees C, respectively). A change in assay temperature significantly decreased surfactant secretion. However, the change in secretory rate between 37 degrees C and 4 degrees C was less than expected if due to temperature alone (Q(10) range=0.8-1.2). Therefore, the surfactant secretory pathway in squirrel type II cells demonstrates some temperature insensitivity. When incubated at the body temperature of the animal from which the cells were isolated, the adrenergic agonist, isoproterenol, significantly increased surfactant secretion in both warm-active and torpid squirrel type II cells. However, the cholinergic agonist, carbamylcholine chloride, only increased secretion in torpid squirrel type II cells when incubated at 4 degrees C. Torpor did not affect basal cAMP production from isolated type II cells. However, the production of cAMP appears to be upregulated in response to isoproterenol in torpid squirrel type II cells. Thus, at the cellular level, both the secretory and regulatory pathways involved in surfactant secretion are thermally insensitive. Upregulating basal secretion and increasing the sensitivity of type II cells to cholinergic stimulation may be adaptative characteristics of torpor that enable type II cells to function effectively at 0-5 degrees C.

The small microchiropteran bat, Chalinolobus gouldii, undergoes large daily fluctuations in metabolic rate, body temperature, and breathing pattern. These alterations are accompanied by changes in surfactant composition, predominantly an increase in cholesterol relative to phospholipid during torpor. Furthermore, the surface activity changes, such that the surfactant functions most effectively at that temperature which matches the animal's activity state. Here, we examine the surface activity of surfactant from bats during arousal from torpor. Bats were housed at 24 degrees C on an 8:16h light:dark cycle and their surfactant was collected during arousal (28

Pulmonary surfactant controls the surface tension at the air-liquid interface within the lung. This system had a single evolutionary origin that predates the evolution of the vertebrates and lungs. The lipid composition of surfactant has been subjected to evolutionary selection pressures, particularly temperature, throughout the evolution of the vertebrates.

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar Type II cells. It reduces alveolar surface tension and hence the work of breathing. Despite the tremendous diversity of lung structures amongst the vertebrates, the composition of surfactant is highly conserved. Conserved elements of the surfactant system amongst distantly related species are likely to be crucial factors for successful lung development. Understanding the mechanisms by which the surfactant system becomes operational in animals with dramatically different birthing strategies and in distantly related species will provide important information about the role of the surfactant system in the commencement of air breathing and the processes regulating surfactant maturation and secretion. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones, and autonomic neurotransmitters. Here we review the mechanisms controlling the maturation of surfactant production, including birthing strategy, phylogeny, lung structure, and posthatching environment. Using four species of egg-laying amniote (chicken, dragon lizard, sea turtle, and crocodile) previously described in detail and the large amount of information available for mammals, we examine the hypothesis that the control of surfactant production is dependent on glucocorticoids (dexamethasone [Dex]), thyroid hormones (T3), and autonomic neurotransmitters (epinephrine and carbachol). We also examine whether the overall intrinsic pattern of the control of surfactant maturation is conserved throughout the vertebrate radiation and then how the environment (extrinsic factors) may account for the observed differences in the patterns of development. We also discuss the utility of a coculture system of embryonic Type II cells and fibroblasts to determine the evolutionary pattern behind the control of surfactant and to demonstrate that the surfactant system matures under multihormonal control. We demonstrate that Dex and T3 are stimulators of surfactant production during embryonic development, but they lose their efficacy closer to hatching or birth. Epinephrine stimulates surfactant secretion beyond 75% of development and also after hatching or birth. Carbachol stimulates surfactant secretion in the bearded dragon and saltwater crocodile but not in the sea turtle, chicken, or mammals. It is likely that the differences in control of surfactant development are likely to be primarily related to metabolic activity and the duration of incubation (i.e., the "speed" of development). Moreover, the hormones examined appear important in promoting development and therefore appear conserved within the amniotes. However, the autonomic neurotransmitters induced different responses in different species. Hence, some factors are crucial for the proper maturation of the surfactant system, whereas others vary throughout evolution without being detrimental to the overall function of the system.

Impaired lymphatic drainage in human limbs causes the debilitating swelling termed lymphoedema. In mammals, known growth factors involved in the control of lymphangiogenesis (growth of new lymph vessels) are vascular endothelial growth factors-C and -D (VEGF-C/D). Here we characterize a model of lymphangiogenesis in which the tail of lizards is regenerated without becoming oedematous. Three weeks after the tail is shed (autotomy), there are a small number of large diameter lymphatic vessels in the regenerated tail. Thereafter, the number increases and the diameter decreases. A functional lymphatic network, as determined by lymphoscintigraphy, is established 6 wk after autotomy. The new network differs morphologically and functionally from that in original tails. This lymphatic regeneration is associated with an up-regulation of a reptilian homologue of the VEGF-C/D protein family (rVEGF-C/D), as determined by Western blot analysis using a human reactive VEGF-C polyclonal antibody. Regenerating lizard tails are potentially useful models for studying the molecular basis of lymphangiogenesis with a view to developing possible treatments for human lymphoedema.

Pulmonary surfactant is a complex mixture of phospholipids (PLs), neutral lipids and proteins that lines the inner surface of the lung. Here it modulates surface tension, thereby increasing lung compliance and preventing the transudation of fluid. In humans, pulmonary surfactant is comprised of approximately 80% PLs, 12% neutral lipids and 8% protein. In most eutherian (i.e. placental) mammals, cholesterol (Chol) comprises approximately 8-10% by weight or 14-20 mol% of both alveolar and lamellar body surfactant. It is regarded as an integral component of pulmonary surfactant, yet few studies have concentrated on its function or control. The lipid composition is highly conserved within the vertebrates, except that surfactant of teleost fish is dominated by cholesterol, whereas tetrapod pulmonary surfactant contains a high proportion of disaturated phospholipids (DSPs). The primitive Australian dipnoan lungfish Neoceratodus forsterii demonstrates a 'fish-type' surfactant profile, whereas the other derived dipnoans demonstrate a surfactant profile similar to that of tetrapods. Homology of the surfactant proteins within the vertebrates points to a single evolutionary origin for the system and indicates that fish surfactant is a 'protosurfactant'. Among the terrestrial tetrapods, the relative proportions of DSPs and cholesterol vary in response to lung structure, habitat and body temperature (Tb), but not in relation to phylogeny. The cholesterol content of surfactant is elevated in species with simple saccular lungs or in aquatic species or in species with low Tb. The DSP content is highest in complex lungs, particularly of aquatic species or species with high Tb. Cholesterol is controlled separately from the PL component in surfactant. For example, in heterothermic mammals (i.e. mammals that vary their body temperature), the relative amount of cholesterol increases in cold animals. The rapid changes in the Chol to PL ratio in response to various physiological stimuli suggest that these two components have different turnover rates and may be packaged and processed differently. In mammals, the pulmonary surfactant system develops towards the end of gestation and is characterized by an increase in the saturation of PLs in lung washings and the appearance of surfactant proteins in amniotic fluid. In general, the pattern of surfactant development is highly conserved among the amniotes. This conservation of process is demonstrated by an increase in the amount and saturation of the surfactant PLs in the final stages (>75%) of development. Although the ratios of surfactant components (Chol, PL and DSP) are remarkably similar at the time of hatching/birth, the relative timing of the maturation of the lipid profiles differs dramatically between species. The uniformity of composition between species, despite differences in lung morphology, birthing strategy and relationship to each other, implies that the ratios are critical for the onset of pulmonary ventilation. The differences in the timing, on the other hand, appear to relate primarily to birthing strategy and the onset of air breathing. As the amount of cholesterol relative to the phospholipids is highly elevated in immature lungs, the pattern of cholesterol during development and evolution represents an example of ontogeny recapitulating phylogeny. The fact that cholesterol is an important component of respiratory structures that are primitive, when they are not in use or developing in an embryo, demonstrates that this substance has important and exciting roles in surfactant. These roles still remain to be explored.

Pulmonary surfactant is a mixture of lipids and proteins that controls the surface tension of the fluid lining the inner lung. Its composition is conserved among the vertebrates. Here we hypothesize that the in ovo administration of glucocorticoids and thyroid hormones during late incubation will accelerate surfactant development in the saltwater crocodile, Crocodylus porosus. We also hypothesize that the increased maturation of the type II cells in response to hormone pretreatment will result in enhanced responsiveness of the cells to surfactant secretagogues. We sampled embryos at days 60, 68, and 75 of incubation and after hatching. We administered dexamethasone (Dex), 3,5,3'-triiodothyronine (T(3)), or a combination of both hormones (Dex + T(3)), 48 and 24 h before each prehatching time point. Lavage analysis indicated that the maturation of the phospholipids (PL) in the lungs of embryonic crocodiles occurs rapidly. Only T(3) and Dex + T(3) increased total PL in lavage at embryonic day 60, but Dex, T(3), and Dex + T(3) increased PL at day 75. The saturation of the PLs was increased by T(3) and Dex + T(3) at day 68. Swimming exercise did not increase the amount or alter the saturation of the surfactant PLs. Pretreatment of embryos with Dex, T(3), or Dex + T(3) changed the secretion profiles of the isolated type II cells. Dex + T(3) increased the response of the cells to agonists at days 60 and 68. Therefore, glucocorticoids and thyroid hormones regulate surfactant maturation in the crocodile.

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar type II cells in the lungs of all air-breathing vertebrates. Pulmonary surfactant functions to reduce the surface tension in the lungs and, therefore, reduce the work of breathing. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones and autonomic neurotransmitters. We have used a co-culture system of embryonic type II cells and lung fibroblasts to investigate the ability of dexamethasone, tri-iodothyronine (T(3)), adrenaline and carbamylcholine (carbachol) to stimulate the cellular secretion of phosphatidylcholine in the bearded dragon (Pogona vitticeps) at day 55 (approx. 92%) of incubation and following hatching. Adrenaline stimulated surfactant secretion both before and after hatching, whereas carbachol stimulated secretion only at day 55. Glucocorticoids and triiodothyronine together stimulated secretion at day 55 but did not after hatching. Therefore, adrenaline, carbachol, dexamethasone and T(3), are all involved in the development of the surfactant system in the bearded dragon. However, the efficacy of the hormones is attenuated during the developmental process. These differences probably relate to the changes in the cellular environment during development and the specific biology of the bearded dragon.

We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T(3)), and a combination of these two hormones (Dex + T(3)). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T(4)) ORD activities in liver and kidney microsomes, and a decrease in the high-K(m) rT(3) ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T(4) ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T(3) IRD activity in liver microsomes. However, this decrease did not change plasma-free T(3) concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development.

The antioxidant enzyme (AOE) system protects the lung from oxidative damage. The pulmonary surfactant (PS) system lowers the interfacial pressure within the lung, improving lung compliance and aiding lung clearance. In mammals, the AOE and PS systems develop in tandem during the final 10%-20% of gestation. Here, we investigated the development of these systems in the viviparous skink, Tiliqua rugosa. The content of total phospholipid (PL), disaturated phospholipid (DSP), and cholesterol (Chol) increased in lung washings from foetal lizards with advancing gestational age. Similarly, the relative saturation of the PLs increased throughout gestation, with mid-stage 40 foetuses having a DSP/PL equivalent to newborns and adults. Maternal lizards had significantly less total PL, DSP, and Chol than nongravid and newborn lizards; however, the relative composition did not differ from nongravid animals. This presumably results from compression of the lungs under the bulk of the developing foetus. The Chol/PL and Chol/DSP ratios declined early in development such that mid-stage 40 embryos had comparable ratios to both newborns and adults. Thus, it appears that the PS system matures in a similar manner in skinks and in mammals. However, the composition of surfactant is complete some weeks before parturition, probably to enable improved survivorship of the precocial young in the event of premature birth. Unlike the surfactant lipids, the AOEs, catalase, superoxide dismutase, and glutathione peroxidase did not differ appreciably throughout gestation. It appears therefore that like the surfactant lipids the AOE system is in readiness for air breathing throughout the latter stages of gestation, possibly in preparation for premature birth. Unlike mammals, the PS and AOE systems develop independently from one another.

The objectives of this survey were to determine the genotoxic potential of sediment pore waters above and below 10 wastewater outfalls and to compare the results to sediment chemical quality guidelines, acute toxicity, and macrobenthic community composition. The focus of the study was on genotoxicity since its occurrence in environmental media below wastewater discharges in the Gulf of Mexico region has not been reported in the scientific literature. Pore waters from 43 sediment samples were assayed using a microbial mutagenicity assay before and after activation with a rat liver microsome mix (S-9). A combination of either direct or activated responses was observed for 40% of the pore waters. Direct, activated, and both direct and activated responses were observed in 5, 26, and 9% of the total samples, respectively. Mutagenic effects were observed below 7 of the 10 outfall areas and in 4 of 6 control areas associated with 6 outfalls. The lowest pore water concentrations causing an activated mutagenic response were statistically similar above and below the outfalls. Mutagenicity occurred more frequently than acute toxicity to estuarine and freshwater invertebrates and there was no consistent relationship between its occurrence and the exceedance of sediment chemical quality guidelines. In contrast, there was some indication that mutagenic activity paralleled low benthic community diversity.

Pulmonary surfactant (PS), a mixture of phospholipids (PL), neutral lipids and surfactant proteins (SP), lowers surface tension within the lung, which increases lung compliance and improves the removal of fluid at birth. Here, we have examined the expression of thyroid transcription factor-1 (TTF-1) and the surfactant protein SP-B, and also the composition of pulmonary surfactant lipids in the developing lung of the turtle Chelydra serpentina. Lavage and lung tissue were collected from late embryonic, pipped and hatchling turtles. TTF-1, a regulator of gene expression of surfactant proteins and cell differentiation in mammals, was detected using immunohistochemistry in epithelia of the gas-exchange area and conducting airways during late development. Expression declined in hatchlings. SP-B was detected in subsets of cells within the respiratory epithelium at all stages sampled. The same cell types also stained for TTF-1. Turtle surfactant lipids matured toward the end of incubation. Maximal secretion of both total phospholipids and disaturated phospholipid (DSP) occurred at the time of pipping, coincident with the onset of breathing. The DSP/PL ratio increased after pipping, whereas cholesterol levels (Chol) increased prior to pipping. This resulted in a decrease in the Chol/PL and Chol/DSP ratios after pipping. Thus, TTF-1 and SP-B appear to be highly conserved within the vertebrates. Maturation of surfactant phospholipid content occurred with the commencement of pulmonary ventilation.

The primary function of pulmonary surfactant is to reduce the surface tension (ST) created at the air-liquid interface in the lung. Surfactant is a complex mixture of lipids and proteins and its function is influenced by physiological parameters such as metabolic rate, body temperature and breathing. In the microchiropteran bat Chalinolobus gouldii these parameters fluctuate throughout a 24 h period. Here we examine the surface activity of surfactant from warm-active and torpid bats at both 24 degrees C and 37 degrees C to establish whether alterations in surfactant composition correlate with changes in surface activity. Bats were housed in a specially constructed bat room at Adelaide University, at 24 degrees C and on a 8:16 h light:dark cycle. Surfactant was collected from bats sampled during torpor (2535 degrees C). Alterations in the lipid composition of surfactant occur with changes in the activity cycle. Most notable is an increase in surfactant cholesterol (Chol) with decreases in body temperature [Codd et al., Physiol. Biochem. Zool. 73 (2000) 605-612]. Surfactant from active bats was more surface active at higher temperatures, indicated by lower ST(min) and less film area compression required to reach ST(min) at 37 degrees C than at 24 degrees C. Conversely, surfactant from torpid bats was more active at lower temperatures, indicated by lower ST(min) and less area compression required to reach ST(min) at 24 degrees C than at 37 degrees C. Alterations in the Chol content of bat surfactant appear to be crucial to allow it to achieve low STs during torpor.

Marsupials are born at an early stage of development and are adapted for future development inside the pouch. Whether the pulmonary surfactant system is fully established at this altricial stage is unknown. This study correlates the presence of surfactant proteins (SP-A, SP-B and SP-D), using immunohistochemistry, with the ex-utero development of the lung in the tammar wallaby Macropus eugenii and also investigates the control of phosphatidylcholine (PC) secretion from developing alveolar type II cells. All three surfactant proteins were found at the site of gas exchange in the lungs of joeys at all ages, even at birth when the lungs are in the early stages of the terminal air-sac phase. Co-cultures of alveolar type II cells and fibroblasts were isolated from the lungs of 30- and 70-day-old joeys and incubated with the hormones dexamethasone (10 micromol l(-1)), prolactin (1 micromol l(-1)) or triiodothyronine (100 micromol l(-1)) or with the autonomic secretagogues isoproterenol (100 micromol l(-1)) or carbamylcholine chloride (100 micromol l(-1)). Basal secretion of PC was greater at 30 days of age than at 70 days. Co-cultures responded to all five agonists at 30 days of age, but only the autonomic secretagogues caused a significant increase in PC secretion at 70 days of age. This demonstrates that, as the cells mature, their activity and responsiveness are reduced. The presence of the surfactant proteins at the site of gas exchange at birth suggests that the system is fully functional. It appears that surfactant development is coupled with the terminal air-sac phase of lung development rather than with birth, the length of gestation or the onset of air-breathing.

The activities of the pulmonary antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase, increase in the final 10-20 % of gestation in the mammalian lung, to protect the lung from attack by increasing levels of reactive oxygen species at birth. Whether the increase occurs as a normal 'preparation for birth', i.e. by a genetically determined mechanism, or in response to increased levels of oxygen, i.e. in response to the environment, is not completely understood. We examined the activities of catalase, SOD and GPx in the developing lungs of two oviparous vertebrate species, the chicken (Gallus gallus) and an agamid lizard (Pogona vitticeps), and in a metamorphosing vertebrate, the anuran Limnodynastes terraereginae. During in ovo development embryos come into contact with higher levels of environmental oxygen, and at a much earlier stage of development, compared with the intrauterine development of mammals. Furthermore, in metamorphosing frogs, the lungs are inflated at an early stage to aid in buoyancy, although the gas-exchange function only develops much later upon final metamorphosis. Here, we hypothesise that the activity of the AOE will be elevated relatively much earlier during development in both oviparous and metamorphosing vertebrates. We also examined the effect of mild hypoxia (17 % oxygen) on the development of the pulmonary AOE in the chicken, to test the hypothesis that these enzymes are responsive to environmental oxygen. In the normoxic lung of both Gallus gallus and Pogona vitticeps, catalase and GPx activities were significantly increased in late incubation, whereas SOD activity decreased in late incubation. Catalase and SOD activities were virtually identical in hypoxic and normoxic embryos of the chicken, but GPx activity was significantly affected by hypoxia. In the developing frog, the activities of all enzymes were high at stage 30, demonstrating that the system is active before the lung displays any significant gas-exchange function. SOD and GPx activity did not increase further with development. Catalase activity increased after stage 40, presumably correlating with an increase in air-breathing. In summary, catalase expression in the two oviparous vertebrates appears to be completely under genetic control as the activity of this enzyme does not change in response to changes in oxygen tension. However, in tadpoles, catalase may be responsive to environmental oxygen. SOD also appears to follow a largely genetically determined program in all species. Under normoxic conditions, GPx appears to follow a genetically determined developmental pattern, but this enzyme demonstrated the largest capacity to respond to environmental oxygen fluctuations. In conclusion, it appears that the AOE are differentially regulated. Furthermore, the AOE in the different species appear to have evolved different levels of dependency on environmental variables. Finally, the late developmental increase in AOE activity seen in mammals is not as pronounced in oviparous and metamorphosing vertebrates.

Pulmonary surfactant, consisting predominantly of phosphatidylcholine (PC), is secreted from Type II cells into the lungs of all air-breathing vertebrates, where it functions to reduce surface tension. In mammals, glucocorticoids and thyroid hormones contribute to the maturation of the surfactant system. It is possible that phylogeny, lung structure, and the environment may influence the development of the surfactant system. Here, we investigate the ontogeny of PC secretion from cocultured Type II cells and fibroblasts in the sea turtle, Chelonia mydas, following 58, 62, and 73 d of incubation and after hatching. The influence of glucocorticoids and thyroid hormones on PC secretion was also examined. Basal PC secretion was lowest at day 58 (3%) and reached a maximal secretion rate of 10% posthatch. Dexamethasone (Dex) alone stimulated PC secretion only at day 58. Triiodothyronine (T(3)) stimulated PC secretion in cells isolated from days 58 and 73 embryos and from hatchling turtles. A combination of Dex and T(3) stimulated PC secretion at all time points.

This study describes the developmental changes in pulmonary surfactant (PS) lipids throughout incubation in the sea turtle, Chelonia mydas. Total phospholipid (PL), disaturated phospholipid (DSP) and cholesterol (Chol) harvested from lung washings increased with advancing incubation, where secretion was maximal at pipping, coincident with the onset of pulmonary ventilation. The DSP/PL ratio increased, whereas the Chol/PL and the Chol/DSP ratio declined throughout development. The phospholipids, therefore, are independently regulated from Chol and their development matches that of mammals. To explore whether hypoxia could elicit an effect on the development of the PS system, embryos were exposed to a chronic dose of 17% O2 for the final approximately 40% of incubation. Hypoxia did not affect incubation time, absolute, nor relative abundance of the surfactant lipids, demonstrating that the development of the system is robust and that embryonic development continues unabated under mild hypoxia. Hypoxia-incubated hatchlings had lighter wet lung weights than those from normoxia, inferring that mild hypoxia facilitates lung clearance in this species.

Pulmonary surfactant (PS) is a complex mixture of phospholipids, neutral lipids and proteins that lines the inner surface of the lung. Here, it modulates surface tension thereby increasing lung compliance and preventing the transudation of fluid. In mammals, the PS system develops towards the end of gestation, characterized by an increase in the saturation of phospholipids in lung washings and the appearance of surfactant proteins in amniotic fluid. Birth, the transition from in utero to the external environment, is a rapid process. At this time, the PS system is important in opening and clearing the lung of fluid in order to initiate pulmonary ventilation. In oviparous vertebrates, escape from an egg can be a long and exhausting process. The young commence pulmonary ventilation and hatching by 'pipping' through the eggshell, where they remain for some time, presumably clearing their lungs. This paper relates changes in the development of the pulmonary surfactant system within the non-mammalian amniotes in response to birth strategy, lung morphology and phylogeny in order to determine the conservatism of this developmental process. Total phospholipid (PL), disaturated phospholipid (DSP) and cholesterol (Chol) were quantified from lung washings of embryonic and hatchling chickens, bearded dragons (oviparous), sleepy lizards (viviparous), snapping turtles and green sea turtles throughout the final stages of incubation and gestation. In all cases, the pattern of development of the pulmonary surfactant lipids was consistent with that of mammals. PL and DSP increased throughout the latter stages of development and Chol was differentially regulated from the PLs. Maximal secretion of both PL and DSP occurred at 'pipping' in oviparous reptiles, coincident with the onset of airbreathing. Similarly, the amount of DSP relative to total PL was maximal immediately after the initiation of airbreathing in chickens. The relative timing of the appearance of the lipids differed between groups. In the oviparous lizard, surfactant lipids were released over a relatively shorter time than that of the sleepy lizard, turtles, birds and mammals. Thus, despite temporal differences and vastly different lung morphologies, birth strategies and phylogenies, the overall development and maturation of the PS system is highly conserved amongst the amniotes.

In most eutherian mammals, cholesterol (Chol) comprises approximately 8-10 wt.% or 14-20 mol.% of both alveolar and lamellar body surfactant. It is regarded as an integral component of pulmonary surfactant, yet few studies have concentrated on its function or control. Throughout the evolution of the vertebrates, the contribution of cholesterol relative to surfactant phospholipids decreases, while that of the disaturated phospholipids (DSP) increases. Chol generally appears to dominate in animals with primitive bag-like lungs that lack septation, in the saccular lung of snakes or swimbladders which are not used predominantly for respiration, and also in immature lungs. It is possible that in these systems, cholesterol represents a protosurfactant. Cholesterol is controlled separately from the phospholipid (PL) component in surfactant. For example, in heterothermic mammals such as the fat-tailed dunnart, Sminthopsis crassicaudata, and the microchiropteran bat, Chalinolobus gouldii, and also in the lizard, Ctenophorus nuchalis, the relative amount of Chol increases in cold animals. During the late stages of embryonic development in chickens and lizards, the Chol to PL and Chol to DSP ratios decrease dramatically. While in isolated lizard lungs, adrenaline and acetylcholine stimulate the secretion of surfactant PL, Chol secretion remains unaffected. This is also supported in isolated cell studies of lizards and dunnarts. The rapid changes in the Chol to PL ratio in response to various physiological stimuli suggest that these two components have different turnover rates and may be packaged and processed differently. Infusion of [3H]cholesterol into the rat tail vein resulted in a large increase in Chol specific activity within 30 min in the lamellar body (LB) fraction, but over a 48-h period, failed to appear in the alveolar surfactant fraction. Analysis of the limiting membrane of the lamellar bodies revealed a high (76%) concentration of LB cholesterol. The majority of lamellar body Chol is, therefore, not released into the alveolar compartment, as the limiting membrane fuses with the cell membrane upon exocytosis. It appears unlikely, therefore, that lamellar bodies are the major source of alveolar Chol. It is possible that the majority of alveolar Chol is synthesised endogenously within the lung and stored independently from surfactant phospholipids. The role of cholesterol in the limiting membrane of the lamellar body may be to enable fast and easy processing by maintaining the membrane in a relatively fluid state.

Richard E. Pattle contributed enormously to the biology of the pulmonary surfactant system. However, Pattle can also be regarded as the founding father of comparative and evolutionary research of the surfactant system. He contributed eight seminal papers of the 167 publications we have located on this topic. In particular, Pattle produced a synthesis interpreting the evolution of the surfactant system that formed the foundation for the area. Prepared 25 years ago this synthesis spawned the three great discoveries in the comparative biology of the surfactant system: (1) that the surfactant system has been highly conserved throughout the enormous radiation of the air breathing vertebrates; (2) that temperature is the major selective condition that influences surfactant composition; (3) that acting as an anti-adhesive is one primitive and ubiquitous function of vertebrate surfactant. Here we review the literature and history of the comparative and evolutionary biology of the surfactant system and highlight the areas of comparative physiology that will contribute to our understanding of the surfactant system in the future. In our view the surfactant system is a neatly packaged system, located in a single cell and highly conserved, yet spectacularly complex. The surfactant system is one of the best systems we know to examine evolutionary processes in physiology as well as gain important insights into gas transfer by complex organisms.