Rodolfo Iturriaga

EVIDENCE A PHOTOPROTECTIVE FOR SECONDARY CAROTENOIDS OF SNOW ALGAE1

Snow algae occupy a unique habitat in high altitude and polar environments. These algae are often subject to extremes in nutrient availability, acidity, solar irradiance, desiccation, and ambient temperature. This report documents the accumulation of secondary carotenoids by snow algae in response to the availability of nitrogenous nutrients. Unusually large accumulations of astaxanthin esters in extra‐chloroplastic lipid globules produce the characteristics red pigmentation typical of some snow algae (e.g. Chlamydomonas nivalis (Bauer) Wille). Consequently these compounds greatly reduce the amount of light available for absorption by the light‐harvesting pigment‐protein complexes, thus potentially limiting photoinhibition and photodamage caused by intense solar radiation. The esterification of astaxnthin with fatty acids represents a possible mechanism by which this chromophore can be concentrated within cytoplasmic globules to maximize its photoprotective efficiency.

MICROALGAL LIGHT-HARVESTING IN EXTREME LOW-LIGHT ENVIRONMENTS IN MCMURDO SOUND, ANTARCTICA1

Microalgal pigment composition, photosynthetic characteristics, single‐cell absorption efficiency (Qa(λ)) spectra, and fluorescence‐excitation (FE) spectra were determined for platelet ice and benthic communities underlying fast ice in McMurdo Sound, Antarctica, during austral spring 1988. Measurements of spectral irradiance (E(λ)) and photosynthetically active radiation (PAR) as well as samples for particulate absorption measurements were taken directly under the congelation ice, within the platelet layer, as profiles vertically through the water column, and at the benihic surface. Light attenuation by.sea ice, algal pigments, and particulates reduced PAR reaching the platelet ice layer to 3%(9–33 fimol photons m‐2‐−s‐1) of surface values and narrowed its spectral distribution to a band between 400 and 580 nm. Attenuation by the water column further reduced PAR reaching the sea floor (28–m depth) to 0.05% of surface levels (< 1 μmol photons m‐2 s‐1), with a spectral distribution dominated by 470–580–nm wavelengths. The photoadaptive index (I) for platelet ice algae (5.9–12.6 μmol photons m‐2.s‐1) was similar to ambient PAR, indicating that algae had acclimated to their light environment (i.e. the algae were light‐replete). Maximum Qa(λ) at the blue absorption peak (440 nm) was 0.63, and enhanced absorption was observed from 460–500 nm and was consistent with observed high cellular chlorophyll (chi) c:chl a and fucoxanthin: chl a molar ratios (0.4 and 1.2, respectively). Benthic algae were light‐limited despite the maintenance of very low Ik values (4–11 μmol photons.m‐2.s‐1). Extremely high fucoxanthin: chi a ratios (1.6) in benthic algae produced enhanced green light absorption, resulting in a high degree of complementation between algal absorption and ambient spectral irradiance. Qa(λ) values for benthic algae were maximal (0.9) between 400 and 510 nm but remained >0.35 even at absorption minima. Strong spectral flattening, a characteristic of intense pigment packaging, was also apparent in the Qa(λ) spectra for benthic algae. FE and Qa(λ) spectra were similar in shape for platelet ice algae, indicating that the efficiency at which absorbed energy was

Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluoresce techniques

The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%. The redox dyes indicated that approximately 4-10% of the cysts were metabolically active 48 h after they were shed, followed by a decline in enzyme activity to near undetectable levels after 4 days. Spectral analysis on individual cysts indicated that the fluorescence emission of the reduced CTC and the fluorescein-labeled antibodies is distinctive for each compound and suitable for their simultaneous determination by microphotometry, flow cytometry and epifluorescence microscopy. The fluorescence signal remained without alteration when the cysts were transferred onto microscope slides coated with an optical embedding medium and stored at -20 degrees C. The fluorescence intensity of the reduced CTC, when properly standardized, can provide quantitative measurements of ETS activity of the cysts. This is the first report of a method to determine enzyme redox activity on intact cysts applicable to water, laboratory and animal samples.

In situ microparticle analysis of marine phytoplankton cells with infrared laser-based optical tweezers

We describe the application of infrared optical tweezers to the in situ microparticle analysis of marine phytoplankton cells. A Nd:YAG laser (λ= 1064 nm) trap is used to confine and manipulate single Nannochloris and Synechococcus cells in an enriched seawater medium while spectral fluorescence and Lorenz-Mie backscatter signals are simultaneously acquired under a variety of excitation and trapping conditions. Variations in the measured fluorescence intensities of chlorophyll a (Chl a) and phycoerythrin pigments in phytoplankton cells are observed. These variations are related, in part, to basic intrasample variability, but they also indicate that increasing ultraviolet-exposure time and infrared trapping power may have short-term effects on cellular physiology that are related to Chl a photobleaching and laser-induced heating, respectively. The use of optical tweezers to study the factors that affect marine cell physiology and the processes of absorption, scattering, and attenuation by individual cells, organisms, and particulate matter that contribute to optical closure on a microscopic scale are also described.

Absorption properties of marine-derived material in Arctic sea ice

The distribution and concentration of particulate and dissolved material were determined for first year and multiyear sea ice collected from the fast ice off Barrow, Alaska. The particles were identified as intact algal cells, bacteria, organic aggregates colonized by bacteria and detrital particles. The largest concentrations of particles occurred in the zones of fine grain or in zones of concentrated brine drainage features. The absorption efficiencies of these particles in combination with measured particle concentrations were shown to provide a reasonable estimate of the bulk particulate spectral absorption coefficients in the case of a first year ice sample. The sum of the absorption coefficients of the particulate and dissolved fractions were up to an order of magnitude greater than the absorption coefficients of pure ice. The enhanced absorption of visible radiation by the entrapped material was estimated to cause increases in sea ice temperature of up to 3 degree(s)C per day in the absence of snow cover. The marine- derived particulate and dissolved materials are not negligible contributors to the optical and thermal characteristics of sea ice.

Influence of particulate matter on spectral irradiance fields and energy transfer in the Eastern Arctic Ocean

Time series studies of the spectral irradiance fields beneath multiyear pack ice were conducted in the Eastern Arctic Basin at 82 - 83 degree(s)N as part of the Coordinated Eastern Arctic Research Experiment (CEAREX). Particulate matter was collected from the multiyear pack ice as well as first year ice in a refrozen lead. The vertical distribution within the ice and the spectral absorption properties of the particulates were determined in order to estimate their contribution to the optical properties of the sea ice. Among the particulates contained in sea ice detritus was common throughout all portions of the pack ice and was the major light absorbing particulate matter in the ice at the time of the observation. Algal cells and mineral- like particulates also were present, yet they contributed to the light-absorbing properties to a lesser extent than the detritus. During early spring, particulate matter contributed little to the bulk attenuation coefficients of the multiyear ice, however, it was estimated to have a more substantial contribution to the attenuation coefficients of first year ice in a refrozen lead. Results of a single stream multilayer radiative transfer model that simulates concentrations of biogenic particulate matter observed in Arctic sea ice indicates that particulate matter within sea ice plays a substantial role in radiative energy transfer and has the potential to seasonally alter spectral irradiance regimes within the ice covered Arctic Ocean.

Individual and Bulk Analysis of the Optical Properties of Marine Particulates: Examples of Merging these Two Scales of Analysis

In recent years cytofluorometric and microphotometric techniques have significantly contributed to the field of biological and optical oceanography. Such determinations have enabled us to better understand the composition of some natural phytoplankton communities. We can now discriminate between Synechococcus clone types in the ocean, sort and analyze minute prochlorophytes, follow microalgae cell cycles, and determine the spectral absorption properties of individual cells as well as detrital particulates of field samples. Analysis at the individual level constitutes another valuable tool for the study of phytoplankton cells and detrital particulates in aquatic systems.

New technique for the determination of spectral reflectance of individual and bulk particulate suspended matter in natural water samples

The behavior of real scattering surfaces is often specified by measuring the bidirectional reflectance factor (BRF), defined as the ratio of the flux scattered into a given direction by a surface under given conditions of illumination to the flux scattered in the same direction by a Lambertian scatterer under identical conditions.THe utility of this factor is that measurements on surfaces can be related to known standards, which have a BRF greater than 99 percent for a broad range of wavelengths. In addition to the incidence angle and spectral features of the incident flux, the reflectance properties of a surface are affected by the intrinsic composition and roughness properties of the surface. Therefore, the spectral reflectance of different targets will generally yield spectral reflectance curves of different shapes, forming the basis for identification of materials. For example, optical principles developed for the determination of reflectance properties of marine particles facilitate the determination of the BRF of oceanic samples. We have recently developed and implemented a system for determining the BRF composed of a Zeiss photomicroscope equipped with a reflective system. In this system, excitation is provided over a large field of view while reflection collection is acquired over a slightly smaller solid angle. Multi-wavelength measurements allow the determination of the effect of the excitation wavelength on both the reflectance and fluorescence properties of the sample, whereas monochromatic measurements exclude fluorescence effects. This new technique provides the advantages of determination of the BRF for different types of individual and bulk particulates transferred onto an optical embedding medium or collected on an Anopore filter. Abundance and other optical properties of dominant particle types can also be determined by individual particle analysis on the same sample.

Preparation of natural phytoplankton communities to preserve spectral fluorescence properties

A technique that allows for the preservation of spectral fluorescence properties of phytoplankton cells is presented. Laboratory cultures and field samples were concentrated and embedded within gelatin to preserve the spectral fluorescence properties of phytoplankton cells. Fluorescence excitation spectra (Ex: 400 - 650 nm, Em: 683 nm) obtained from samples prepared according to this method were compared to the spectra obtained from traditional cell suspensions, and by microphotometry. The spectra produced by samples prepared following this method remain spectrally intact if stored at -70 degree(s)C, and allowed to return to room temperature prior to measurement.

Long-Term Preservation of Microalgal Cells and their Optical Properties

A unique protocol using a gelatin-based embedding technique allows long-term preservation of sea ice microalgae and phytoplankton cells. The high quality preservation of the cells and their optical properties for over two decades was confirmed after a re-examination of samples collected and prepared during 1987 at McMurdo Station, Antarctica and during 1990 at the California Bight near Los Angeles. Samples stored frozen until 2011 demonstrated the long-term preservation of the cellular structure, as well as their spectral absorption and fluorescence properties. This protocol makes it possible to assemble archives of sea ice microalgae and phytoplankton cells for environmental studies.