Fusarium: more than a node or a foot-shaped basal cell.

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

Genome-scale phylogenies reveal relationships among Parastagonospora species infecting domesticated and wild grasses.

Several plant pathogenic Parastagonospora species have been identified infecting wheat and other cereals over the past 50 years. As new lineages were discovered, naming conventions grew unwieldy and the relationships with previously recognized species remained unclear. We used genome sequencing to clarify relationships among these species and provided new names for most of these species. Six of the nine described Parastagonospora species were recovered from wheat, with five of these species coming from Iran. Genome sequences revealed that three strains thought to be hybrids between P. nodorum and P. pseudonodorum were not actually hybrids, but rather represented rare gene introgressions between those species. Our data are consistent with the hypothesis that P. nodorum originated as a pathogen of wild grasses in the Fertile Crescent, then emerged as a wheat pathogen via host-tracking during the domestication of wheat in the same region. The discovery of a diverse array of Parastagonospora species infecting wheat in Iran suggests that new wheat pathogens could emerge from this region in the future. Citation: Croll D, Crous PW, Pereira D, et al. 2021. Genome-scale phylogenies reveal relationships among Parastagonospora species infecting domesticated and wild grasses. Persoonia 46: 116-128. https://doi.org/10.3767/persoonia.2021.46.04.

Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation.

UNLABELLED: The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis. IMPORTANCE: Fungi cause life-threatening diseases, but very few drugs are available to effectively treat fungal infections. The pathogenic fungus Cryptococcus neoformans causes a substantial global burden of life-threatening meningitis in patients suffering from HIV/AIDS. An understanding of the mechanisms by which fungi deploy virulence factors to cause disease is critical for developing new therapeutic approaches. We employed a quantitative proteomic approach to define the changes in the protein complement that occur upon modulating the cAMP signaling pathway that regulates virulence in C. neoformans. This approach identified a conserved role for cAMP signaling in the regulation of the ubiquitin-proteasome pathway and revealed a link between this pathway and elaboration of a major virulence determinant, the polysaccharide capsule. Targeting the ubiquitin-proteasome pathway opens new therapeutic options for the treatment of cryptococcosis.

QTL mapping of temperature sensitivity reveals candidate genes for thermal adaptation and growth morphology in the plant pathogenic fungus Zymoseptoria tritici.

Different thermal environments impose strong, differential selection on populations, leading to local adaptation, but the genetic basis of thermal adaptation is poorly understood. We used quantitative trait locus (QTL) mapping in the fungal wheat pathogen Zymoseptoria tritici to study the genetic architecture of thermal adaptation and identify candidate genes. Four wild-type strains originating from the same thermal environment were crossed to generate two mapping populations with 263 (cross 1) and 261 (cross 2) progeny. Restriction site-associated DNA sequencing was used to genotype 9745 (cross 1) and 7333 (cross 2) single-nucleotide polymorphism markers segregating within the mapping population. Temperature sensitivity was assessed using digital image analysis of colonies growing at two different temperatures. We identified four QTLs for temperature sensitivity, with unique QTLs found in each cross. One QTL had a logarithm of odds score >11 and contained only six candidate genes, including PBS2, encoding a mitogen-activated protein kinase kinase associated with low temperature tolerance in Saccharomyces cerevisiae. This and other QTLs showed evidence for pleiotropy among growth rate, melanization and growth morphology, suggesting that many traits can be correlated with thermal adaptation in fungi. Higher temperatures were highly correlated with a shift to filamentous growth among the progeny in both crosses. We show that thermal adaptation has a complex genetic architecture, with natural populations of Z. tritici harboring significant genetic variation for this trait. We conclude that Z. tritici populations have the potential to adapt rapidly to climate change and expand into new climatic zones.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads.

In sharp contrast to birds and mammals, most cold-blooded vertebrates have homomorphic (morphologically undifferentiated) sex chromosomes. This might result either from recurrent X-Y recombination (occurring e.g. during occasional events of sex reversal) or from frequent turnovers (during which sex-determining genes are overthrown by new autosomal mutations). Evidence for turnovers is indeed mounting in fish, but very few have so far been documented in amphibians, possibly because of practical difficulties in identifying sex chromosomes. Female heterogamety (ZW) has long been established in Bufo bufo, based on sex reversal and crossing experiments. Here, we investigate a sex-linked marker identified from a laboratory cross between Palearctic green toads (Bufo viridis subgroup). The F(1) offspring produced by a female Bufo balearicus and a male Bufo siculus were phenotypically sexed, displaying an even sex ratio. A sex-specific marker detected in highly reproducible AFLP genotypes was cloned. Sequencing revealed a noncoding, microsatellite-containing fragment. Reamplification and genotyping of families of this and a reciprocal cross showed B. siculus to be male heterogametic (XY) and suggested the same system for B. balearicus. Our results thus reveal a cryptic heterogametic transition within bufonid frogs and help explain patterns of hybrid fitness within the B. viridis subgroup. Turnovers of genetic sex-determination systems may be more frequent in amphibians than previously thought and thus contribute to the prevalence of homomorphic sex chromosomes in this group.

Introduced predators transform subarctic islands from grassland to tundra.

Top predators often have powerful direct effects on prey populations, but whether these direct effects propagate to the base of terrestrial food webs is debated. There are few examples of trophic cascades strong enough to alter the abundance and composition of entire plant communities. We show that the introduction of arctic foxes (Alopex lagopus) to the Aleutian archipelago induced strong shifts in plant productivity and community structure via a previously unknown pathway. By preying on seabirds, foxes reduced nutrient transport from ocean to land, affecting soil fertility and transforming grasslands to dwarf shrub/forb-dominated ecosystems.

Immune function of cryopreserved avian peripheral white blood cells: potential biomarkers of contaminant effects in wild birds.

Contaminants can cause detrimental effects in wild birds. However, these effects are difficult to measure in all but the most severe cases. Immune function is a sensitive and meaningful biological marker of contaminant-induced effects in captive birds but has more limitations in wild birds due in part to the lack of a proven blood preservation method. We developed methods to assess ex vivo immune function in wild birds using cryopreserved peripheral white blood cells (WBCs). We assessed the effects of cryopreservation on WBC viability and functionality in two immunoassays (concavalin A-induced T lymphocyte proliferation and macrophage phagocytosis) in domestic chickens (Gallus spp.: white Wyandottes and Dominiques) and validated this approach on cryopreserved WBC samples from wild American coots (Fulicia americana). Cryopreservation of chicken WBCs caused a slight but significant decrease in cell viability (99% +/- 0.2 SE for fresh cells versus 84% +/- 2 SE for cryopreserved cells, p = 0.001, Mann-Whitney U, n = 8). No difference was detected in viability between cells that were cryopreserved for less than 10 days (88% +/- 3.7 SE) and more than 50 days (89% +/- 1.3 SE) (n = 6). Overall, there was no statistical difference in the performance of cryopreserved cells compared to fresh cells. Across multiple experiments, cryopreserved T lymphocytes exhibited 200-900% stimulated proliferation above nonstimulated cells, and 40-80% of cryopreserved macrophages ingested yeast. 9,10,Dimethyl-1,2-benz-anthracene (DMBA) reduced proliferation and phagocytosis in cryopreserved cells over an ex vivo exposure range of 0-170 microM DMBA. Tests of immune function on American coot WBCs cryopreserved for up to 10 months (viability of 72% +/- 2.5 SE, n = 24) were similar to the cryopreserved chicken WBCs. This study will facilitate greater use of ex vivo immune function assays as tools to study effects of contaminant exposure in wildlife by demonstrating the viability and functionality of cryopreserved avian cells.

High feeding costs limit dive time in the largest whales.

Large body size usually extends dive duration in air-breathing vertebrates. However, the two largest predators on earth, the blue whale (Balaenoptera musculus) and the fin whale (B. physalus), perform short dives for their size. Here, we test the hypothesis that the foraging behavior of these two species (lunge-feeding) is energetically expensive and limits their dive duration. We estimated the cost of lunge-feeding in both species using an approach that combined attaching time/depth recorders to seven blue whales and eight fin whales and comparing the collected dive information with predictions made by optimality models of dive behavior. We show that the rate at which whales recovered from a foraging dive was twice that of a non-foraging dive and that the cost of foraging relative to the cost of travel to and from the prey patch was 3.15 in blue whales (95 % CI 2.58-3.72) and 3.60 in fin whales (95 % CI 2.35-4.85). Whales foraged in small areas (<1 km(2)) and foraging bouts lasted more than one dive, indicating that prey did not disperse and thus that prey dispersal could not account for the limited dive durations of the whales. Despite the enormous size of blue whales and fin whales, the high energetic costs of lunge-feeding confine them to short durations of submergence and to areas with dense prey aggregations. As a corollary, because of their limited foraging time under water, these whales may be particularly vulnerable to perturbations in prey abundance.

The diving behavior of blue and fin whales: is dive duration shorter than expected based on oxygen stores?

Many diving seabirds and marine mammals have been found to regularly exceed their theoretical aerobic dive limit (TADL). No animals have been found to dive for durations that are consistently shorter than their TADL. We attached time-depth recorders to 7 blue whales and 15 fin whales (family Balaenopteridae). The diving behavior of both species was similar, and we distinguished between foraging and traveling dives. Foraging dives in both species were deeper, longer in duration and distinguished by a series of vertical excursions where lunge feeding presumably occurred. Foraging blue whales lunged 2.4 (+/-1.13) times per dive, with a maximum of six times and average vertical excursion of 30.2 (+/-10.04) m. Foraging fin whales lunged 1.7 (+/-0.88) times per dive, with a maximum of eight times and average vertical excursion of 21.2 (+/-4.35) m. The maximum rate of ascent of lunges was higher than the maximum rate of descent in both species, indicating that feeding lunges occurred on ascent. Foraging dives were deeper and longer than non-feeding dives in both species. On average, blue whales dived to 140.0 (+/-46.01) m and 7.8 (+/-1.89) min when foraging, and 67.6 (+/-51.46) m and 4.9 (+/-2.53) min when not foraging. Fin whales dived to 97.9 (+/-32.59) m and 6.3 (+/-1.53) min when foraging and to 59.3 (+/-29.67) m and 4.2 (+/-1.67) min when not foraging. The longest dives recorded for both species, 14.7 min for blue whales and 16.9 min for fin whales, were considerably shorter than the TADL of 31.2 and 28.6 min, respectively. An allometric comparison of seven families diving to an average depth of 80-150 m showed a significant relationship between body mass and dive duration once Balaenopteridae whales, with a mean dive duration of 6.8 min, were excluded from the analysis. Thus, the short dive durations of blue whales and fin whales cannot be explained by the shallow distribution of their prey. We propose instead that short duration diving in large whales results from either: (1) dispersal behavior of prey; or (2) a high energetic cost of foraging.

Sink or swim: strategies for cost-efficient diving by marine mammals.

Locomotor activity by diving marine mammals is accomplished while breath-holding and often exceeds predicted aerobic capacities. Video sequences of freely diving seals and whales wearing submersible cameras reveal a behavioral strategy that improves energetic efficiency in these animals. Prolonged gliding (greater than 78% descent duration) occurred during dives exceeding 80 meters in depth. Gliding was attributed to buoyancy changes with lung compression at depth. By modifying locomotor patterns to take advantage of these physical changes, Weddell seals realized a 9.2 to 59.6% reduction in diving energetic costs. This energy-conserving strategy allows marine mammals to increase aerobic dive duration and achieve remarkable depths despite limited oxygen availability when submerged.

Mechanical versus physiological determinants of swimming speeds in diving Brünnich's guillemots.

For fast flapping flight of birds in air, the maximum power and efficiency of the muscles occur over a limited range of contraction speeds and loads. Thus, contraction frequency and work per stroke tend to stay constant for a given species. In birds such as auks (Alcidae) that fly both in air and under water, wingbeat frequencies in water are far lower than in air, and it is unclear to what extent contraction frequency and work per stroke are conserved. During descent, compression of air spaces dramatically lowers buoyant resistance, so that maintaining a constant contraction frequency and work per stroke should result in an increased swimming speed. However, increasing speed causes exponential increases in drag, thereby reducing mechanical versus muscle efficiency. To investigate these competing factors, we have developed a biomechanical model of diving by guillemots (Uria spp.). The model predicted swimming speeds if stroke rate and work per stroke stay constant despite changing buoyancy. We compared predicted speeds with those of a free-ranging Brünnich's guillemot (U. lomvia) fitted with a time/depth recorder. For descent, the model predicted that speed should gradually increase to an asymptote of 1.5-1.6 m s-1 at approximately 40 m depth. In contrast, the instrumented guillemot typically reached 1.5 m s-1 within 10 m of the water surface and maintained that speed throughout descent to 80 m. During ascent, the model predicted that guillemots should stroke steadily at 1.8 m s-1 below their depth of neutral buoyancy (62 m), should alternate stroking and gliding at low buoyancies from 62 to 15 m, and should ascend passively by buoyancy alone above 15 m depth. However, the instrumented guillemot typically ascended at 1.25 m s-1 when negatively buoyant, at approximately 1.5 m s-1 from 62 m to 25 m, and supplemented buoyancy with stroking above 25 m. Throughout direct descent, and during ascent at negative and low positive buoyancies (82-25 m), the guillemot maintained its speed within a narrow range that minimized the drag coefficient. In films, guillemots descending against high buoyancy at shallow depths increased their stroke frequency over that of horizontal swimming, which had a substantial glide phase. Model simulations also indicated that stroke duration, relative thrust on the downstroke versus the upstroke, and the duration of gliding can be varied to regulate swimming speed with little change in contraction speed or work per stroke. These results, and the potential use of heat from inefficient muscles for thermoregulation, suggest that diving guillemots can optimize their mechanical efficiency (drag) with little change in net physiological efficiency.

Validation of deuterium incorporation against sterol balance for measurement of human cholesterol biosynthesis.

To examine the validity of the deuterium (D) incorporation technique for measurement of human cholesterol synthesis rates, D uptake from D2O into cholesterol was compared to sterol balance in 13 subjects each under three controlled diet settings. Subjects (age 62 +/- 3.6 yr, body weight 74 +/- 4.0 kg, BMI 27 +/- 1.4) consumed weight maintenance diets enriched in either corn oil, beef tallow, or stick corn oil margarine over a 5-week period. During the final week of the study period, subjects were given 1.2 g/D2O per kg body water. D enrichment was measured in plasma water and total cholesterol over 24 h. Also, during the final week, dietary intake and fecal elimination rates of cholesterol were assessed over one 6-day period to calculate sterol balance. There was no significant difference (t = 0.858, P = 0.397) between D incorporation into cholesterol (1,183 +/- 92 mg/day) and sterol balance (1,316 +/- 125 mg/day). Among diets, net cholesterol biosynthesis measured by D incorporation agreed (r = 0.745, P = 0.0001) with values derived from sterol balance. The degree of association between methods was not influenced by the wide range of fatty acid composition of the diet fat. These data demonstrate the utility of the simple, non-restrictive deuterium incorporation method as a reliable means of determining cholesterol biosynthesis in free-living humans.

Cholesterol metabolism in New World primates: comparative studies in two tamarin species (Saguinus oedipus and Saguinus fuscicollis) and the squirrel monkey (Saimiri sciureus).

1. Cholesterol metabolism has been characterized in three species of New World primates, the cotton-top tamarin, the saddle-back tamarin, and the squirrel monkey. 2. When fed a diet containing cholesterol, the three species exhibited differing responses of plasma cholesterol levels. 3. Dietary cholesterol absorption was determined and plasma cholesterol die-away kinetics were analyzed in terms of a two-pool model. 4. The results of the analyses of cholesterol turnover are consistent with the observed species-specific differences in plasma cholesterol values and cholesterol absorption. 5. Cholesterol metabolism differs between the two tamarin species, as well as between the tamarins and the squirrel monkey. 6. Implications of species-specific differences between tamarin species are discussed in terms of the use of tamarin species as animal models for comparative studies of cholesterol metabolism and the etiology of cancer and cardiovascular disease.

Diving metabolism and thermoregulation in common and thick-billed murres.

The diving and thermoregulatory metabolic rates of two species of diving seabird, common (Uria aalge) and thick-billed murres (U. lomvia), were studied in the laboratory. Post-absorptive resting metabolic rates were similar in both species, averaging 7.8 W.kg-1, and were not different in air or water (15-20 degrees C). These values were 1.5-2 times higher than values predicted from published allometric equations. Feeding led to increases of 36 and 49%, diving caused increases of 82 and 140%, and preening led to increases of 107 and 196% above measured resting metabolic rates in common and thick-billed murres, respectively. Metabolic rates of both species increased linearly with decreasing water temperature; lower critical temperature was 15 degrees C in common murres and 16 degrees C in thick-billed murres. Conductance (assuming a constant body temperature) did not change with decreasing temperature, and was calculated at 3.59 W.m-2 x degrees C-1 and 4.68 W.m-2 x degrees C-1 in common and thick-billed murres, respectively. Murres spend a considerable amount of time in cold water which poses a significant thermal challenge to these relatively small seabirds. If thermal conductance does not change with decreasing water temperature, murres most likely rely upon increasing metabolism to maintain body temperature. The birds probably employ activities such as preening, diving, or food-induced thermogenesis to meet this challenge.

The effect of submergence on heart rate and oxygen consumption of swimming seals and sea lions.

Respiratory, metabolic, and cardiovascular responses to swimming were examined in two species of pinniped, the harbor seal (Phoca vitulina) and the California sea lion (Zalophus californianus). 1. Harbor seals remained submerged for 82-92% of the time at swimming speeds below 1.2 m.s-1. At higher speeds, including simulated speeds above 1.4 m.s-1, the percentage of time spent submerged decreased, and was inversely related to body weight. In contrast, the percentage of time spent submerged did not change with speed for sea lions swimming from 0.5 m.s-1 to 4.0 m.s-1. 2. During swimming, harbor seals showed a distinct breathhold bradycardia and ventilatory tachycardia that were independent of swimming speed. Average heart rate was 137 beats.min-1 when swimming on the water surface and 50 beats.min-1 when submerged. A bimodal pattern of heart rate also occurred in sea lions, but was not as pronounced as in the seals. 3. The weighted average heart rate (WAHR), calculated from measured heart rate and the percentage time spent on the water surface or submerged, increased linearly with swimming speed for both species. The graded increase in heart rate with exercise load is similar to the response observed for terrestrial mammals. 4. The rate of oxygen consumption increased exponentially with swimming speed in both seals and sea lions. The minimum cost of transport calculated from these rates ranged from 2.3 to 3.6 J.m-1.kg-1, and was 2.5-4.0 times the level predicted for similarly-sized salmonids. Despite different modes of propulsion and physiological responses to swimming, these pinnipeds demonstrate similar transport costs.

Calcium wave evoked by activation of endogenous or exogenously expressed receptors in Xenopus oocytes.

The mRNA encoding the cloned substance K receptor was microinjected into Xenopus laevis oocytes. After expression of the mRNA, Ca2+ was imaged in the oocytes with a digital imaging fluorescence microscopy system using the Ca2(+)-sensitive dyes fura-2 and fluo-3. Application of substance K caused a dose-related wave of Ca2+ mobilization to spread from a focus and to elevate the Ca2+ concentration in the oocyte. Activation of endogenous muscarinic or angiotensin II receptors in noninjected oocytes evoked a similar response. The Ca2+ rise in oocytes induced by substance K was due to internal Ca2+ mobilization and was independent of external Ca2+, since it occurred in Ca2(+)-free medium fortified with 2 mM EGTA. The Ca2+ imaging was well correlated with ion current measurements of voltage-clamped oocytes. Imaging, in addition to detecting the spatial spread of Ca2+ across the cell, was at least as sensitive as voltage clamping and much faster when screening oocytes for the expression of receptor mRNAs that stimulate Ca2+ mobilization. While it is known that fertilization of Xenopus eggs causes a spreading wave of Ca2+ mobilization, we found that activation of either native or newly expressed receptors in oocytes causes a similar change in Ca2+ distribution.

Cardiac output and stroke volume in swimming harbor seals.

Cardiac output was measured by the thermodilution method in three young harbor seals, at rest and while swimming up to the maximum effort for which they could be trained. Stroke volume was determined by counting heart rate simultaneously with determination of cardiac output. Cardiac outputs varied widely between surface breathing (7.8 ml.kg-1.s-1) and breath-holding while swimming under water (1.8 ml.kg-1.s-1). Stroke volume while at the surface was almost twice the volume while submerged. Surface cardiac output was always near maximal despite work effort, whereas submerged cardiac output gradually increased at higher work efforts. The cardiovascular performance of seals at the maximum MO2 we could induce from them is equivalent to that of the domestic goat.