Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Acanthamoebal surface properties and the modulation of phagocytosis

The surface nature of Acanthamoeba trophozoites and cysts was investigated with respect to cell surface charge, hydrophobicity and surface carbohydrate composition. Particulate microelectrophoresis revealed a marked negative charge for both morphological forms, though less for cyst surfaces. Hydrophobicity was determined by adhesion to n-hexadecane and indicated a relatively low hydrophobic nature of both forms, though less so for cysts. Surface carbohydrate composition was studied by the use of fluorescent lectins and flow cytometry, using a ligand-receptor approach for further in depth analysis of binding of particular lectins. These studies showed trophozoite and cyst surfaces to be rich in N-acetylglucosamine, N-acteylneuraminic acid, mannose and glucose, with the addition of N-acetylgalactosamine on cysts. The importance of such surface properties was investigated with respect to phagocytosis of polystyrene latex microspheres, of different surface types and size. Investigations into the optimum conditions of uptake of beads indicated a preference for a medium devoid of nutrients, such as saline, though temperature was not a factor. An amoebal predilection for beads of lower charge and greater hydrophobicity was demonstrated. Furthermore, a preference for the largest bead size used (2.0 m) was observed. The influence of either Con A or mannose or glucose on bead association was apparently limited. The fate of foreign DNA ingested by Acanthamoeba appeared to indicate that such DNA was destroyed, as it could not be detected following extraction procedures and PCR amplification.

Some clinical aspects of the eye in extended contact lens wear

The limbal vascular response to extended contact lens wear was examined in a group comparative study initially intended to last eighteen months. After six months all patients wearing contact lenses had presented with micro-epithelial cysts. This unanticipated occurrence of the micro-epithelial-cysts necessitated termination of the study, and limited the quantity of data collected. However, sufficient results were available to allow a limited description of •the vascular response to this form of contact lens wear. Interpretations of the date collected ore discussed in relation to suggested vasostimulating factors in the cornea. The micro-epithelial cysts observed after extended wear were classified and their rate of recovery recorded. A further clinical study was undertaken to observe cysts in both contact lens - and non contact lens-wearing eyes. Cysts were observed in every category of patient, although the characteristic patterns varied. These observations of micro-epithelial cysts are discussed with respect to the aetiopathogeneses of corneal epithelial cystic disorders. Subsequently, attempts were made to induce cysts in rabbit corneae by extended contact lens wear. Clinical observations revealed cyst-like appearances. Histological sections did not contain cysts but did exhibit signs characteristic •of cystic disorders of the corneal epithelium. In general, the results from the study indicate that extended wear is subjectively acceptable to contact lens wearers. However, the objective findings of significant vascular changes, micro-epithelial cysts and cases of acute red eye response cast considerable doubt on the recommendation of extended wear contact lenses for purely cosmetic applications.

Spectrum of morphological and visual changes due to vitreomacular interface disorders encountered in a large consecutive cohort of patients

Aim: Identify the incidence of vitreomacular traction (VMT) and frequency of reduced vision in the absence of other coexisting macular pathology using a pragmatic classification system for VMT in a population of patients referred to the hospital eye service. Methods: A detailed survey of consecutive optical coherence tomography (OCT) scans was done in a high-throughput ocular imaging service to ascertain cases of vitreomacular adhesion (VMA) and VMT using a departmental classification system. Analysis was done on the stages of traction, visual acuity, and association with other macular conditions. Results: In total, 4384 OCT scan episodes of 2223 patients were performed. Two hundred and fourteen eyes had VMA/VMT, with 112 eyes having coexisting macular pathology. Of 102 patients without coexisting pathology, 57 patients had VMT grade between 2 and 8, with a negative correlation between VMT grade and number of Snellen lines (r= -0.61717). There was a distinct cutoff in visual function when VMT grade was higher than 4 with the presence of cysts and sub retinal separation and breaks in the retinal layers. Conclusions: VMT is a common encounter often associated with other coexisting macular pathology. We estimated an incidence rate of 0.01% of VMT cases with reduced vision and without coexisting macular pathology that may potentially benefit from intervention. Grading of VMT to select eyes with cyst formation as well as hole formation may be useful for targeting patients who are at higher risk of visual loss from VMT.

(Table 1) Elemental mean values of the upper Paleocene and lower Eocene in IODP Hole 302-M0004A

We reconstruct the latest Paleocene and early Eocene (~57-50 Ma) environmental trends in the Arctic Ocean and focus on the Paleocene-Eocene thermal maximum (PETM) (~55 Ma), using strata recovered from the Lomonosov Ridge by the Integrated Ocean Drilling Program Expedition 302. The Lomonosov Ridge was still partially subaerial during the latest Paleocene and earliest Eocene and gradually subsided during the early Eocene. Organic dinoflagellate cyst (dinocyst) assemblages point to brackish and productive surface waters throughout the latest Paleocene and early Eocene. Dinocyst assemblages are cosmopolitan during this time interval, suggesting warm conditions, which is corroborated by TEX86'-reconstructed temperatures of 15°-18°C. Inorganic geochemistry generally reflects reducing conditions within the sediment and euxinic conditions during the upper lower Eocene. Spectral analysis reveals that the cyclicity, recorded in X-ray fluorescence scanning Fe data from close to Eocene thermal maximum 2 (~53 Ma, presence confirmed by dinocyst stratigraphy), is related to precession. Within the lower part of the PETM, proxy records indicate enhanced weathering, runoff, anoxia, and productivity along with sea level rise. On the basis of total organic carbon content and variations in sediment accumulation rates, excess organic carbon burial in the Arctic Ocean appears to have contributed significantly to the sequestration of injected carbon during the PETM.

Absolute abundances of dinoflagellate cysts in surface sediment samples from four sites: Lycopodium marker-grain method

Absolute abundances (concentrations) of dinoflagellate cysts are often determined through the addition of Lycopodium clavatum marker-grains as a spike to a sample before palynological processing. An inter-laboratory calibration exercise was set up in order to test the comparability of results obtained in different laboratories, each using its own preparation method. Each of the 23 laboratories received the same amount of homogenized splits of four Quaternary sediment samples. The samples originate from different localities and consisted of a variety of lithologies. Dinoflagellate cysts were extracted and counted, and relative and absolute abundances were calculated. The relative abundances proved to be fairly reproducible, notwithstanding a need for taxonomic calibration. By contrast, excessive loss of Lycopodium spores during sample preparation resulted in non-reproducibility of absolute abundances. Use of oxidation, KOH, warm acids, acetolysis, mesh sizes larger than 15 µm and long ultrasonication (> 1 min) must be avoided to determine reproducible absolute abundances. The results of this work therefore indicate that the dinoflagellate cyst worker should make a choice between using the proposed standard method which circumvents critical steps, adding Lycopodium tablets at the end of the preparation and using an alternative method.

(Appendix B) Dinoflagellate cysts in Southeast Pacific Ocean surface sediments

orty-eight surface sediment samples from the southeast (SE) Pacific (25-53°S) are investigated for the determination of the spatial distribution of organic-walled dinoflagellate cysts along the western South American continental margin. Fifty-five different taxa are recorded and reflect oceanic or coastal assemblages. The oceanic assemblages are characterised by low cyst concentrations and the dominance of autotrophs, while the coastal assemblages generally contain a higher number of cysts, which are mainly produced by heterotrophic species. Highest cyst concentrations are observed in the active upwelling system offshore Concepción (35-37°S). Brigantedinium spp., Echinidinium aculeatum, Echinidinium granulatum/delicatum and cysts of Protoperidinium americanum dominate assemblages related to upwelling. Echinidinium aculeatum appears to be the best indicator for the presence of all year round active upwelling cells. Other protoperidinioid cysts may also occur in high relative abundances in coastal regions outside active upwelling systems, if the availability of nutrients, co-responsible for the presence/absence of their main food sources such as diatoms and other protists, is sufficient. The importance of nutrient availability as a determining environmental variable influencing cyst signals on a regional scale (SE Pacific) is demonstrated through statistical analyses of the data. Because of the importance of nutrients, uncertainties about the outcomes of quantitative sea-surface temperature (SST) reconstructions (Modern Analogue Technique) based on dinoflagellate cysts may arise, since no interaction between different hydrographical variables is considered in this approach. The combination of the SE Pacific surface sample dataset with other published cyst data from the Southern Hemisphere resulted in a database which includes 350 samples: the 'SH350 database'. This database is used to test the accuracy of the quantitative reconstructions by calculating and comparing the estimated versus observed values for each site. An attempt to perform quantitative SST reconstructions on the last 25 cal ka of site ODP1233 (41°S; 74°27'W) is made and again stresses the importance of other environmental variables such as nutrient availability in determining the dinoflagellate cyst assemblages.

Relative abundance of dinoflagellate cysts in surface sediments and dinoflagellate counts from three sediment cores from the Atlantic sector of the Southern Ocean

In this study we investigate the potential of organic-walled dinoflagellate cysts (dinocysts) as tools for quantifying past sea-surface temperatures (SST) in the Southern Ocean. For this purpose, a dinocyst reference dataset has been formed, based on 138 surface sediment samples from different circum-Antarctic environments. The dinocyst assemblages of these samples are composed of phototrophic (gonyaulacoid) and heterotrophic (protoperidinioid) species that provide a broad spectrum of palaeoenvironmental information. The relationship between the environmental parameters in the upper water column and the dinocyst distribution patterns of individual species has been established using the statistical method of Canonical Correspondence Analysis (CCA). Among the variables tested, summer SST appeared to correspond to the maximum variance represented in the dataset. To establish quantitative summer SST reconstructions, a Modern Analogue Technique (MAT) has been performed on data from three Late Quaternary dinocyst records recovered from locations adjacent to prominent oceanic fronts in the Atlantic sector of the Southern Ocean. These dinocyst time series exhibit periodic changes in the dinocyst assemblage during the last two glacial/interglacial-cycles. During glacial conditions the relative abundance of protoperidinioid cysts was highest, whereas interglacial conditions are characterised by generally lower cyst concentrations and increased relative abundance of gonyaulacoid cysts. The MAT palaeotemperature estimates show trends in summer SST changes following the global oxygen isotope signal and a strong correlation with past temperatures of the last 140,000 years based on other proxies. However, by comparing the dinocyst results to quantitative estimates of summer SSTs based on diatoms, radiolarians and foraminifer-derived stable isotope records it can be shown that in several core intervals the dinocyst-based summer SSTs appeared to be extremely high. In these intervals the dinocyst record seems to be highly influenced by selective degradation, leading to unusual temperature ranges and to unrealistic palaeotemperatures. We used the selective degradation index (kt-index) to determine those intervals that have been biased by selective degradation in order to correct the palaeotemperature estimates. We show that after correction the dinocyst based SSTs correspond reasonably well with other palaeotemperature estimates for this region, supporting the great potential of dinoflagellate cysts as a basis for quantitative palaeoenvironmental studies.

Dinocyst-derived sea-surface characteristics, palynomorph abundances, ice-rafted debris, and planktonic foraminifera abundances and d18O of the 35-41 ka BP interval of core MD99-2285

The last glacial millennial climatic events (i.e. Dansgaard-Oeschger and Heinrich events) constitute outstanding case studies of coupled atmosphere-ocean-cryosphere interactions. Here, we investigate the evolution of sea-surface and subsurface conditions, in terms of temperature, salinity and sea ice cover, at very high-resolution (mean resolution between 55 and 155 years depending on proxies) during the 35-41 ka cal BP interval covering three Dansgaard-Oeschger cycles and including Heinrich event 4, in a new unpublished marine record, i.e. the MD99-2285 core (62.69°N; -3.57s°E). We use a large panel of complementary tools, which notably includes dinocyst-derived sea-ice cover duration quantifications. The high temporal resolution and multiproxy approach of this work allows us to identify the sequence of processes and to assess ocean-cryosphere interactions occurring during these periodic ice-sheet collapse events. Our results evidence a paradoxical hydrological scheme where (i) Greenland interstadials are marked by a homogeneous and cold upper water column, with intensive winter sea ice formation and summer sea ice melting, and (ii) Greenland and Heinrich stadials are characterized by a very warm and low saline surface layer with iceberg calving and reduced sea ice formation, separated by a strong halocline from a less warm and saltier subsurface layer. Our work also suggests that this stadial surface/subsurface warming started before massive iceberg release, in relation with warm Atlantic water advection. These findings thus support the theory that upper ocean warming might have triggered European ice-sheet destabilization. Besides, previous paleoceanographic studies conducted along the Atlantic inflow pathways close to the edge of European ice-sheets suggest that such a feature might have occurred in this whole area. Nonetheless, additional high resolution paleoreconstructions are required to confirm such a regional scheme.

Temporal variation of temperature and related proxies from the penultimate glacial towards the Eemian in the Black Sea

The last glacial-interglacial transition or Termination I (T I) is well documented in the Black Sea, whereas little is known about climate and environmental dynamics during the penultimate Termination (T II). Here we present a multi-proxy study based on a sediment core from the SE Black Sea covering the penultimate glacial and almost the entire Eemian interglacial (133.5 ±0.7-122.5 ±1.7 ka BP). Proxies comprise ice-rafted debris (IRD), O and Sr isotopes as well as Sr/Ca, Mg/Ca, and U/Ca ratios of benthic ostracods, organic and inorganic sediment geochemistry, as well as TEX86 and UK'37derived water temperatures. The ending penultimate glacial (MIS 6, 133.5 to 129.9 ±0.7 ka BP) is characterised by mean annual lake surface temperatures of about 9°C as estimated from the TEX86 palaeothermometer. This period is impacted by two Black Sea melt water pulses (BSWP-II-1 and 2) as indicated by very low Sr/Ca ostracods but high sedimentary K/Al values. Anomalously high radiogenic 87Sr/86Sr ostracod values (max. 0.70945) during BSWP-II-2 suggest a potential Himalayan source communicated via the Caspian Sea. The T II warming started at 129.9 ±0.7 ka BP, witnessed by abrupt disappearance of IRD, increasing d18O ostracod values, and a first TEX86 derived temperature rise of about 2.5°C. A second, abrupt warming step to ca. 15.5°C as the prelude of the Eemian warm period is documented at 128.3 ka BP. The Mediterranean-Black Sea reconnection most likely occurred at 128.1 ±0.7 ka BP as demonstrated by increasing Sr/Ca ostracods and U/Ca ostracods values. The disappearance of ostracods and TOC contents >2% document the onset of Eemian sapropel formation at 127.6 ka BP. During sapropel formation, TEX86 temperatures dropped and stabilised at around 9°C, while UK'37 temperatures remain on average 17°C. This difference is possibly caused by a habitat shift of Thaumarchaeota communities from surface towards nutrient-rich deeper and colder waters located above the gradually establishing halo-and redoxcline.

Dinoflagellate cysts in surface sediments

Surface sediments at 439 sites throughout the North Atlantic Ocean and adjacent seas have been analyzed for dinoflagellate cysts in order to establish a reference database from which paleoenvironmental transfer functions can be developed. Laboratory procedures and systematics were standardized in order to avoid bias introduced by the selective loss of taxa and to facilitate site to site comparison. 371 sites were retained to develop the database that includes 41 taxa, some of which were grouped using morphological and/or ecological criteria. 27 taxa were retained for statistical purposes. Distribution maps of these latter taxa have been plotted on the basis of their relative abundance. Principal component analyses were performed in order to describe the distribution of assemblages. The relation between the assemblages, as well as the relative abundance of individual taxa, and selected sea-surface parameters are illustrated. The parameters which were considered include temperature and salinity for winter (February) and summer (August) together with the duration of sea-ice cover. Transfer functions using the best analogue method have been tested with a view to reconstruct past sea-surface parameters. Validation procedures on this transfer function demonstrate that more than 95% of the reconstructions are included within the interannual variability of modern sea-surface conditions. Therefore, these transfer functions give accurate results and can be applied to reconstructing paleo-temperatures and -salinities from analogous assemblages in Quaternary sedimentary sequences. Protoperidinium stellatum (Wall in Wall & Dale, 1968) Head, comb. nov. (basionym = Peridinium stellatum) is proposed as new, and Algidasphaeridium? minutum var. cezare de Vernal et al., 1989 ex de Vernal et al. is newly validated.

Abundance of organic-walled dinoflagellate cysts in surface sediments from the Atlantic sector of the Southern Ocean (Appendix 2)

Thirty-two surface sediment samples from the Southern Ocean (eastern Atlantic sector), between the Subtropical Front and the Weddell Gyre, were investigated to provide information on the distribution of modern organic-walled dinoflagellate cysts in relation to the oceanic fronts of the Antarctic Circumpolar Current (ACC). A clearly distinguishable distribution pattern was observed in relation to the water masses and fronts of the ACC. The dinoflagellate cysts of species characteristic of open oceanic environments, such as Impagidinium species, are highly abundant around the Subtropical Front, whereas south of this front, cosmopolitan species such as Nematosphaeropsis labyrinthus and the cysts of Protoceratium reticulatum characterise the transition from subtropical to subantarctic surface waters. The subantarctic surface waters are dominated by the cysts of heterotrophic dinoflagellates, such as Protoperidinium spp. and Selenopemphix antarctica. The cysts of Protoperidinium spp. form the dominant part of the assemblages around the Antarctic Polar Front, whereas S. antarctica concentrations increase further to the south. The presence of S. antarctica in sediments of the Maud Rise, a region of seasonal sea-ice cover, reflects its tolerance for low temperatures and sea-ice cover. A previously undescribed species, Cryodinium meridianum gen. nov. sp. nov., has a restricted distribution pattern between the Antarctic Polar Front and the ACC-Weddell Gyre Boundary.

Grain-size, lithic grains, foraminifera-derived and dinocyst-derived data of sediment core MD99-2281

The last glacial period was punctuated by abrupt climatic events with extrema known as Heinrich and Dansgaard-Oeschger events. These millennial events have been the subject of many paleoreconstructions and model experiments in the past decades, but yet the hydrological processes involved remain elusive. In the present work, high-resolution analyses were conducted on the 12-42 ka BP section of core MD99-2281 retrieved southwest of the Faeroe Islands, and combined with analyses conducted in two previous studies (Zumaque et al., 2012; Caulle et al., 2013). Such a multiproxy approach, coupling micropaleontological, geochemical and sedimentological analyses, allows us to track surface, subsurface, and deep hydrological processes occurring during these rapid climatic changes. Records indicate that the coldest episodes of the studied period (Greenland stadials and Heinrich stadials) were characterized by a strong stratification of surface waters. This surface stratification seems to have played a key role in the dynamics of subsurface and deep-water masses. Indeed, periods of high surface stratification are marked by a coupling of subsurface and deep circulations which sharply weaken at the beginning of stadials, while surface conditions progressively deteriorate throughout these cold episodes; conversely, periods of decreasing surface stratification (Greenland interstadials) are characterized by a coupling of surface and deep hydrological processes, with progressively milder surface conditions and gradual intensification of the deep circulation, while the vigor of the subsurface northward Atlantic flow remains constantly high. Our results also reveal different and atypical hydrological signatures during Heinrich stadials (HSs): while HS1 and HS4 exhibit a "usual" scheme with reduced overturning circulation, a relatively active North Atlantic circulation seems to have prevailed during HS2, and HS3 seems to have experienced a re-intensification of this circulation during the middle of the event. Our findings thus bring valuable information to better understand hydrological processes occurring in a key area during the abrupt climatic shifts of the last glacial period.