Disentangling the role of light and nutrient limitation on bacterivory by mixotrophic nanoflagellates.

Many phytoplankton taxa function on multiple trophic levels by combining photosynthesis and ingestion of bacteria, termed mixotrophy. Despite the recognition of mixotrophy as a universal functional trait, we have yet to fully resolve how environmental conditions influence community grazing rates in situ. A microcosm study was used to assess bacterivory by mixotrophic nanoflagellates following nutrient enrichment and light attenuation in a temperate lake. We found contrasting results based on assessment of mixotroph abundance or bacterivory. Despite an interactive effect of nutrient enrichment and light attenuation on mixotroph abundance, significant differences within light treatments were observed only after enrichment with P or N + P. The greatest abundance of mixotrophs across treatments occurred under co-nutrient enrichment with full exposure to irradiance. However, bacterivory by mixotrophic nanoflagellates was greatest under shaded conditions after either N or P enrichment. We suggest that PAR availability dampened the stimulatory effect of nutrient limitation, and bacterivory supplemented a suboptimal photosynthetic environment. In a saturating light regime, the mixotrophic community was less driven to ingest bacteria because photosynthesis was able to satisfy energetic demands. These findings quantify community bacterivory in response to environmental drivers that may characterize future ecosystem conditions and highlight the importance of considering grazing rates in conjunction with abundance of mixotrophic protists.

Effects of Temperature and Photorepair Radiation on a Marine Ciliate Exposed to UVB Radiation.

The influences of intensity and repeated exposure to ultraviolet-B radiation (UVB), photoreactivating repair radiation (PRR), and temperature on the scuticociliate Parauronema acutum were explored under laboratory conditions. Population growth was negatively affected after exposure to the equivalent of one sunny summer day of ambient UVB, especially in the absence of PRR. Repeated daily exposure to UVB severely compromised ciliate survival. UVB-exposed treatments without PRR recovered slower and reached lower final abundances than treatments receiving PRR. Reducing the daily UVB exposure approximately 25% improved ciliate recovery after exposure. In the single exposure treatments, temperature effects were not consistent, except that growth was slowest for control and treatments at the lowest temperature (15 °C). These data suggest that dark repair and/or photoprotection are present in P. acutum, but photoenzymatic repair was the more effective mechanism in reversing UVB damage. Repeated exposure treatments without PRR had zero or declining growth at all temperatures (15, 20 and 25 °C), as did those with PRR at 15 °C. Significant temperature/dose differences were identified in the repeated exposure treatments; ciliates subjected to the higher UVB intensity with PRR survived only at 25 °C, while ciliate populations under reduced UVB increased at 20 and 25 °C.

Oxygen exposures at NASA's Neutral Buoyancy Lab: a 20-year experience.

Astronauts training for extravehicular activity (EVA) operations can spend many hours submerged underwater in a pressurized suit, called an extravehicular mobility unit (EMU), exposed to pressures exceeding 2 atmospheres absolute (ATA). To minimize the risk of decompression sickness (DCS) a 46% nitrox mixture is used. This limits the nitrogen partial pressure, decreasing the risk of DCS. The trade-off with using a 46% nitrox mixture is the increased potential for oxygen toxicity, which can lead to severe neurologic symptoms including seizures. Suited runs, which typically expose astronauts of 0.9-1.1 ATA for longer than six hours, routinely exceed the recommendation for central nervous system oxygen toxicity limits (CNSOTL) published by the National Oceanic and Atmospheric Administration (NOAA). Fortunately, in over 50,000 hours of suited training dives spanning 20 years of EVA training operations at NASA's Neutral Buoyancy Laboratory (NBL) there has never been an occurrence of oxygen toxicity. This lends support to anecdotal sentiment among certain members of the hyperbaric community that the NOAA CNSOTL recommendations might be overly conservative, at least for the oxygen pressure and time regime in which NBL operates. The NOAA CNSOTL recommendations are the result of expert consensus with a focus on safety and do not necessarily reflect rigorous experimental evidence. The data from the NBL suited dive operations provide a foundation of evidence that can help inform the expert discussion on dive-related neurologic oxygen toxicity performance and overnight recovery in young, healthy males.

Hypertension screening of commercial divers from an occupational medicine perspective.

Hypertension is one of the most common chronic diseases in the world: in the United States, it affects one-third of the adult population. Most cases are primary hypertension - with no identified cause - and significantly increase risk for cardiovascular disease and stroke. At NASA's Neutral Buoyancy Laboratory (NBL), there is a cadre of professional divers who provide underwater support during extravehicular activity training (EVAs, commonly referred to as "spacewalks") for astronauts. The NBL occupational health program requires regular "work-site" physical exams, including blood pressure checks, on at least a weekly basis. Although some blood pressures measured during such regular checks were elevated, the diagnosis and management of blood pressure was outside the scope of that occupational medicine program. As a result, there was no protocol to analyze and manage asymptomatic elevated blood pressure if and when it warranted physician follow-up. We therefore analyzed the blood pressure data from all divers in 2015, vetted the worksite physical and developed a referral pathway to ensure divers have access to effective management of their blood pressure while minimizing impact of such a program on daily operations.

Temperature-dependent phagotrophy and phototrophy in a mixotrophic chrysophyte.

The roles of temperature and light on grazing and photosynthesis were examined for Dinobryon sociale, a common freshwater mixotrophic alga. Photosynthetic rate was determined for D. sociale adapted to temperatures of 8, 12, 16, and 20°C under photosynthetically active radiation light irradiances of 25, 66, and 130 µmol photons · m(-2)  · s(-1) , with concurrent measurement of bacterial ingestion at all temperatures under medium and high light (66 and 130 µmol photons · m(-2)  · s(-1) ). Rates of ingestion and photosynthesis increased with temperature to a maximum at 16°C under the two higher light regimes, and declined at 20°C. Although both light and temperature had a marked effect on photosynthesis, there was no significant difference in bacterivory at medium and high irradiances at any given temperature. At the lowest light condition (25 µmol photons · m(-2)  · s(-1) ), photosynthesis remained low and relatively stable at all temperatures. D. sociale acquired the majority of carbon from photosynthesis, although the low photosynthetic rate without a concurrent decline in feeding rate at 8°C suggested 20%-30% of the carbon budget could be attributed to bacterivory at low temperatures. Grazing experiments in nutrient-modified media revealed that this mixotroph had increased ingestion rates when either dissolved nitrogen or phosphorus was decreased. This work increases our understanding of environmental effects on mixotrophic nutrition. Although the influence of abiotic factors on phagotrophy and phototrophy in pure heterotrophs and phototrophs has been well studied, much less is known for mixotrophic organisms.

Physiological responses of three species of Antarctic mixotrophic phytoflagellates to changes in light and dissolved nutrients.

Antarctic phototrophs are challenged by extreme temperatures, ice cover, nutrient limitation, and prolonged periods of darkness. Yet this environment may also provide niche opportunities for phytoplankton utilizing alternative nutritional modes. Mixotrophy, the combination of photosynthesis and particle ingestion, has been proposed as a mechanism for some phytoplankton to contend with the adverse conditions of the Antarctic. We conducted feeding experiments using fluorescent bacteria-sized tracers to compare the effects of light and nutrients on bacterivory rates in three Antarctic marine photosynthetic nanoflagellates representing two evolutionary lineages: Cryptophyceae (Geminigera cryophila) and Prasinophyceae (Pyramimonas tychotreta and Mantoniella antarctica). Only G. cryophila had previously been identified as mixotrophic. We also measured photoautotrophic abilities over a range of light intensities (P vs. I) and used dark survival experiments to assess cell population dynamics in the absence of light. Feeding behavior in these three nanoflagellates was affected by either light, nutrient levels, or a combination of both factors in a species-specific manner that was not conserved by evolutionary lineage. The different responses to environmental factors by these mixotrophs supported the idea of tradeoffs in the use of phagotrophy and phototrophy for growth.

Selective feeding and foreign plastid retention in an Antarctic dinoflagellate.

The peridinin-containing plastid found in most photosynthetic dinoflagellates is thought to have been replaced in a few lineages by plastids of chlorophyte, diatom, or haptophyte origin. Other distinct lineages of phagotrophic dinoflagellates retain functional plastids obtained from algal prey for different durations and with varying source species specificity. 18S rRNA gene sequence analyses have placed a novel gymnodinoid dinoflagellate isolated from the Ross Sea (RSD) in the Kareniaceae, a family of dinoflagellates with permanent plastids of haptophyte origin. In contrast to other species in this family, the RSD contains kleptoplastids sequestered from its prey, Phaeocystis antarctica. Culture experiments were employed to determine whether the RSD fed selectively on P. antarctica when offered in combination with another polar haptophyte or cryptophyte species, and whether the RSD, isolated from its prey and starved, would take up plastids from P. antarctica or from other polar haptophyte or cryptophyte species. Evidence was obtained for selective feeding on P. antarctica, plastid uptake from P. antarctica, and increased RSD growth in the presence of P. antarctica. The presence of a peduncle-like structure in the RSD suggests that kleptoplasts are obtained by myzocytosis. RSD cells incubated without P. antarctica were capable of survival for at least 29.5 months. This remarkable longevity of the RSD's kleptoplasts and its species specificity for prey and plastid source is consistent with its prolonged co-evolution with P. antarctica. It may also reflect the presence of a plastid protein import mechanism and genes transferred to the dinokaryon from a lost permanent haptophyte plastid.

Intracapsular algae provide fixed carbon to developing embryos of the salamander Ambystoma maculatum.

Each spring, North American spotted salamander (Ambystoma maculatum) females each lay hundreds of eggs in shallow pools of water. Eggs are surrounded by jelly layers and are deposited as large gelatinous masses. Following deposition, masses are penetrated by a mutualistic green alga, Oophila amblystomatis, which enters individual egg capsules, proliferates and aggregates near the salamander embryo, providing oxygen that enhances development. We examined the effects of population density of intracapsular O. amblystomatis on A. maculatum embryos and show that larger algal populations promote faster embryonic growth and development. Also, we show that carbon fixed by O. amblystomatis is transferred to the embryos, providing the first evidence of direct translocation of photosynthate from a symbiont to a vertebrate host.

Mixoplankton and mixotrophy: future research priorities.

Phago-mixotrophy, the combination of photoautotrophy and phagotrophy in mixoplankton, organisms that can combine both trophic strategies, have gained increasing attention over the past decade. It is now recognized that a substantial number of protistan plankton species engage in phago-mixotrophy to obtain nutrients for growth and reproduction under a range of environmental conditions. Unfortunately, our current understanding of mixoplankton in aquatic systems significantly lags behind our understanding of zooplankton and phytoplankton, limiting our ability to fully comprehend the role of mixoplankton (and phago-mixotrophy) in the plankton food web and biogeochemical cycling. Here, we put forward five research directions that we believe will lead to major advancement in the field: (i) evolution: understanding mixotrophy in the context of the evolutionary transition from phagotrophy to photoautotrophy; (ii) traits and trade-offs: identifying the key traits and trade-offs constraining mixotrophic metabolisms; (iii) biogeography: large-scale patterns of mixoplankton distribution; (iv) biogeochemistry and trophic transfer: understanding mixoplankton as conduits of nutrients and energy; and (v) in situ methods: improving the identification of in situ mixoplankton and their phago-mixotrophic activity.

Seizure during hyperbaric oxygen therapy for carbon monoxide toxicity: a case series and five-year experience.

BACKGROUND: Hyperbaric oxygen (HBO) therapy is recommended to reduce the delayed neurologic sequelae resulting from carbon monoxide (CO) toxicity. Although HBO is generally well tolerated, there exists a risk of seizure in all patients that may be increased in patients with predisposing factors including: fever, hypothermia, prior seizure, or brain injury. CASE REPORT: We present two cases of patients without known risk factors who experienced seizures associated with HBO therapy during treatment for CO toxicity. CONCLUSION: This facility's 5-year experience and a review of the germane literature are also presented to elucidate the risk factors and incidence of seizures in patients treated with HBO for CO toxicity.

Diversity of Microbial Eukaryotes Along the West Antarctic Peninsula in Austral Spring.

During a cruise from October to November 2019, along the West Antarctic Peninsula, between 64.32 and 68.37°S, we assessed the diversity and composition of the active microbial eukaryotic community within three size fractions: micro- (> 20 µm), nano- (20-5 µm), and pico-size fractions (5-0.2 µm). The communities and the environmental parameters displayed latitudinal gradients, and we observed a strong similarity in the microbial eukaryotic communities as well as the environmental parameters between the sub-surface and the deep chlorophyll maximum (DCM) depths. Chlorophyll concentrations were low, and the mixed layer was shallow for most of the 17 stations sampled. The richness of the microplankton was higher in Marguerite Bay (our southernmost stations), compared to more northern stations, while the diversity for the nano- and pico-plankton was relatively stable across latitude. The microplankton communities were dominated by autotrophs, mostly diatoms, while mixotrophs (phototrophs-consuming bacteria and kleptoplastidic ciliates, mostly alveolates, and cryptophytes) were the most abundant and active members of the nano- and picoplankton communities. While phototrophy was the dominant trophic mode, heterotrophy (mixotrophy, phagotrophy, and parasitism) tended to increase southward. The samples from Marguerite Bay showed a distinct community with a high diversity of nanoplankton predators, including spirotrich ciliates, and dinoflagellates, while cryptophytes were observed elsewhere. Some lineages were significantly related-either positively or negatively-to ice coverage (e.g., positive for Pelagophyceae, negative for Spirotrichea) and temperature (e.g., positive for Cryptophyceae, negative for Spirotrichea). This suggests that climate changes will have a strong impact on the microbial eukaryotic community.

Alternative nutritional strategies in protists: symposium introduction and a review of freshwater protists that combine photosynthesis and heterotrophy.

The alternative nutritional strategies in protists that were addressed during the symposium by that name at the 2010 annual meeting of the International Society of Protistologists and here in contributed papers, include a range of mechanisms that combine photosynthesis with heterotrophy in a single organism. Often called mixotrophy, these multiple trophic level combinations occur across a broad range of organisms and environments. Consequently, there is great variability in the physiological abilities and relative importance of phototrophy vs. phagotrophy and/or osmotrophy in mixotrophic protists. Recently, research papers addressing ecological questions about mixotrophy in marine systems have been more numerous than those that deal with freshwater systems, a trend that is probably partly due to a realization that many harmful algal blooms in coastal marine systems involve mixotrophic protists. After an introduction to the symposium presentations, recent studies of mixotrophy in freshwater systems are reviewed to encourage continuing research on their importance to inland waters.

Direct effects of UV-B radiation on the freshwater heterotrophic nanoflagellate Paraphysomonas sp.

The formation of DNA photoproducts in organisms exposed to ambient levels of UV-B radiation can lead to death and/or reduced population growth in aquatic systems. Dependence on photoenzymatic repair to reverse DNA damage caused by UV-B radiation is demonstrated for Paraphysomonas sp., a member of a widely distributed genus of heterotrophic nanoflagellates. At 20 degrees C, Paraphysomonas sp. was exposed to a range of UV-B intensities encountered in natural systems. Populations of the flagellate survived and grew in a dose-dependent manner, but only when simultaneously exposed to photorepair radiation (PRR). In contrast, flagellates exposed to UV-B at 15 degrees C suffered 100% mortality except at the lowest UV-B level (with PRR) tested, which suggested a photorepair temperature optimum above 15 degrees C. After acute UV-B exposures, DNA damage (measured as the formation of pyrimidine dimers) was reduced only in organisms that underwent subsequent exposure to PRR. Populations kept in the dark after UV-B exposure maintained the initial levels of pyrimidine dimers. These results are the first to demonstrate the reliance of a heterotrophic flagellate on photoenzymatic DNA repair for survival from UV-B exposure.

Carbon Monoxide Levels in the Extravehicular Mobility Unit by Modeling and Operational Testing.

INTRODUCTION: Carbon monoxide (CO) is a toxic gas with potential for detriment to spaceflight operations. An analytical model was developed to investigate if a maximum CO contamination of 1 ppm in the oxygen (O2) supply reached dangerous levels during extravehicular activity (EVA). Occupational monitoring pre- and postsuited exposures provided supplementary data for review.METHODS: The analytical model estimated O2 and CO concentrations in the extravehicular mobility unit (EMU) based on O2 and CO flow rates into and out of the system. The model was based on 3 h of prebreathe at 15.2 psia, 8 h of EVA at 4.3 psia, and 1 h at 15.2 psia for suit doffing. The Coburn-Forster-Kane equation was used to calculate crewmember carboxyhemoglobin saturation (COHb%) as a function of time. Monitoring of hemoglobin CO saturation (Spco) with a CO-oximeter was conducted pre- and post-EVA during operations on the International Space Station and in ground-based analog environments.RESULTS: The model predicted a maximum PCO in the EMU of 0.061 mmHg and a maximum crewmember COHb% of 2.1%. Operational Spco measurements in mean ± SD during ground-based analog testing were 0.7% ± 1.8% pretest and 0.5% ± 1.5% posttest. Spco values on the ISS were 1.5% ± 0.7% pre-EVA and 1.1% ± 0.3% post-EVA.DISCUSSION: The model predicted that astronauts are not exposed to toxic levels of CO during EVA and operational measurements did not show significant differences between Spco levels between pre- and post-EVA.Makowski MS, Norcross JR, Alexander D, Sanders RW, Conkin J, Young M. Carbon monoxide levels in the extravehicular mobility unit by modeling and operational testing. Aerosp Med Hum Perform. 2019; 90(2):84-91.

A Systematic Review and Meta-Analysis of Decompression Sickness in Altitude Physiological Training.

INTRODUCTION: A review of decompression sickness (DCS) cases associated with the NASA altitude physiological training (APT) program at the Johnson Space Center (JSC) motivated us to place our findings into the larger context of DCS prevalence from other APT centers.METHODS: We reviewed JSC records from 1999 to 2016 and 14 publications from 1968 to 2004 about DCS prevalence in other APT programs. We performed a meta-analysis of 15 APT profiles (488 cases / 385,116 exposures). We used meta-regression to evaluate the relation between estimated exposures and probability of DCS in a test group, accounting for the heterogeneity between studies.RESULTS: Our in-house review identified 6 Type I DCS (1 from an inside observer) and 1 Type II DCS. There were 6 cases in 9560 student hypobaric exposures from 3 NASA training flights; a student pooled prevalence rate of 0.44 cases / 1000 exposures compared to 1.44 cases / 1000 from 12 published APT profiles. The overall pooled DCS prevalence rate was 1.16 cases / 1000 exposures. There was substantial heterogeneity in DCS prevalence across studies. Denitrogenation time, exposure pressure, and exposure time were associated with probability of DCS in the meta-regression model.CONCLUSIONS: While the overall DCS prevalence rate is relatively low, there is marked heterogeneity among profiles. The pooled DCS prevalence rate estimate for the NASA profiles was lower than the overall rate. Variability in APT profile DCS prevalence could be further explained given student level and additional test-level covariates.Conkin J, Sanders RW, Koslovsky MD, Wear ML, Kozminski AG, Abercromby AFJ. A systematic review and meta-analysis of decompression sickness in altitude physiological training. Aerosp Med Hum Perform. 2018; 89(11):941-951.

Phagotrophy by the picoeukaryotic green alga Micromonas: implications for Arctic Oceans.

Photosynthetic picoeukaryotes (PPE) are recognized as major primary producers and contributors to phytoplankton biomass in oceanic and coastal environments. Molecular surveys indicate a large phylogenetic diversity in the picoeukaryotes, with members of the Prymnesiophyceae and Chrysophyseae tending to be more common in open ocean waters and Prasinophyceae dominating coastal and Arctic waters. In addition to their role as primary producers, PPE have been identified in several studies as mixotrophic and major predators of prokaryotes. Mixotrophy, the combination of photosynthesis and phagotrophy in a single organism, is well established for most photosynthetic lineages. However, green algae, including prasinophytes, were widely considered as a purely photosynthetic group. The prasinophyte Micromonas is perhaps the most common picoeukaryote in coastal and Arctic waters and is one of the relatively few cultured representatives of the picoeukaryotes available for physiological investigations. In this study, we demonstrate phagotrophy by a strain of Micromonas (CCMP2099) isolated from Arctic waters and show that environmental factors (light and nutrient concentration) affect ingestion rates in this mixotroph. In addition, we show size-selective feeding with a preference for smaller particles, and determine P vs I (photosynthesis vs irradiance) responses in different nutrient conditions. If other strains have mixotrophic abilities similar to Micromonas CCMP2099, the widespread distribution and frequently high abundances of Micromonas suggest that these green algae may have significant impact on prokaryote populations in several oceanic regimes.

Photosynthetic carbon from algal symbionts peaks during the latter stages of embryonic development in the salamander Ambystoma maculatum.

BACKGROUND: It was recently discovered that symbiotic algae in the eggs of the salamander Ambystoma maculatum translocate fixed carbon from photosynthesis to developing embryos. Fixed carbon translocation was shown in embryos at one time point during development, however, it was unknown if fixed carbon translocation occurs throughout all developmental stages. FINDINGS: In this study, fixed carbon translocation was measured in salamander eggs at six time points over the latter half of development. Fixed carbon translocation did not occur until the middle tailbud portion of development (stages 26-30), and translocation was measured in 20% or less of eggs sampled. Peak carbon translocation occurred during the late tailbud phase of development (stages 31-35), where as much as 87% of eggs sampled showed translocation, and average percent translocation was 6.5%. During the final stages of development, fixed carbon translocation declined, and translocation was not detected in embryos five days prior to hatching. CONCLUSIONS: The onset of fixed carbon translocation from Oophila to A. maculatum embryos during the second half of embryonic development is likely due to the corresponding settlement and concentration of Oophila in the inner egg envelope. In addition, carbon translocation ceases in late stage embryos as the inner egg envelope thins and ruptures in preparation for hatching.

Antarctic mixotrophic protist abundances by microscopy and molecular methods.

Protists are traditionally described as either phototrophic or heterotrophic, but studies have indicated that mixotrophic species, organisms that combine both strategies, can have significant impacts on prey populations in marine microbial food webs. While estimates of active mixotroph abundances in environmental samples are determined microscopically by fluorescent particle ingestion, species identification is difficult. We developed SYBR-based qPCR strategies for three Antarctic algal species that we identified as mixotrophic. This method and traditional ingestion experiments were applied to determine the total mixotroph abundance in Antarctic water samples, to ascertain the abundance of known mixotrophic species, and to identify environmental variables that impact the distribution and abundance of these species. Despite differences in sampling locations and years, mixotroph distribution was strongly influenced by season. Environmental variables that best explained variation in the individual mixotroph species abundances included temperature, oxygen, date, fluorescence, conductivity, and latitude. Phosphate was identified as an additional explanatory variable when nutrients were included in the analysis. Utilizing culture-based grazing rates and qPCR abundances, the estimated summed impact on bacterial populations by the three mixotrophs was usually < 2% of the overall mixotrophic grazing, but in one sample, Pyramimonas was estimated to contribute up to 80% of mixotrophic grazing.

Bacterivory by phototrophic picoplankton and nanoplankton in Arctic waters.

Mixotrophy, the combination of phototrophy and heterotrophy within the same individual, is widespread in oceanic systems. Yet, neither the presence nor ecological impact of mixotrophs has been identified in an Arctic marine environment. We quantified nano- and picoplankton during early autumn in the Beaufort Sea and Canada Basin and determined relative rates of bacterivory by heterotrophs and mixotrophs. Results confirmed previous reports of low microbial biomass for Arctic communities in autumn. The impact of bacterivory was relatively low, ranging from 0.6 × 10(3) to 42.8 × 10(3) bacteria mL(-1) day(-1) , but it was often dominated by pico- or nanomixotrophs. From 1% to 7% of the photosynthetic picoeukaryotes were bacterivorous, while mixotrophic nanoplankton abundance comprised 1-22% of the heterotrophic and 2-32% of the phototrophic nanoplankton abundance, respectively. The estimated daily grazing impact was usually < 5% of the bacterial standing stock, but impacts as high as 25% occurred. Analysis of denaturing gradient gel electrophoresis (DGGE) band patterns indicated that communities from different depths at the same site were appreciably different and that there was a shift in community diversity at the midpoint of the cruise. Sequence information from DGGE bands reflected microbes related to those from other Arctic studies, particularly from the Beaufort Sea.