Metaproteomics of marine viral concentrates reveals key viral populations and abundant periplasmic proteins in the oligotrophic deep chlorophyll maximum of the South China Sea.

Viral concentrates (VCs), containing bioinformative DNA and proteins, have been used to study viral diversity, viral metagenomics and virus-host interactions in natural ecosystems. Besides viruses, VCs also contain many noncellular biological components including diverse functional proteins. Here, we used a shotgun proteomic approach to characterize the proteins of VCs collected from the oligotrophic deep chlorophyll maximum (DCM) of the South China Sea. Proteins of viruses infecting picophytoplankton, that is, cyanobacteria and prasinophytes, and heterotrophic bacterioplankton, such as SAR11 and SAR116, dominated the viral proteome. Almost no proteins from RNA viruses or known gene transfer agents were detected, suggesting that they were not abundant at the sampling site. Remarkably, nonviral proteins made up about two thirds of VC proteins, including overwhelmingly abundant periplasmic transporters for nutrient acquisition and proteins for diverse cellular processes, that is, translation, energy metabolism and one carbon metabolism. Interestingly, three 56 kDa selenium-binding proteins putatively involved in peroxide reduction from gammaproteobacteria were abundant in the VCs, suggesting active removal of peroxide compounds at DCM. Our study demonstrated that metaproteomics provides a valuable avenue to explore the diversity and structure of the viral community and also the pivotal biological functions affiliated with microbes in the natural environment.

Land classification and change intensity analysis in a coastal watershed of Southeast China.

The aim of this study is to improve the understanding of land changes in the Jiulong River watershed, a coastal watershed of Southeast China. We developed a stratified classification methodology for land mapping, which combines linear stretching, an Iterative Self-Organizing Data Analysis (ISODATA) clustering algorithm, and spatial reclassification. The stratified classification for 2002 generated less overall error than an unstratified classification. The stratified classifications were then used to examine temporal differences at 1986, 1996, 2002, 2007 and 2010. Intensity Analysis was applied to analyze land changes at three levels: time interval, category, and transition. Results showed that land use transformation has been accelerating. Woodland's gains and losses were dormant while the gains and losses of Agriculture, Orchard, Built-up and Bare land were active during all time intervals. Water's losses were active and stationary. The transitions from Agriculture, Orchard, and Water to Built-up were systematically targeting and stationary, while the transition from Woodland to Built-up was systematically avoiding and stationary.

Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05), and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

Detecting the dynamic linkage between landscape characteristics and water quality in a subtropical coastal watershed, Southeast China.

Geospatial analysis and statistical analysis are coupled in this study to determine the dynamic linkage between landscape characteristics and water quality for the years 1996, 2002, and 2007 in a subtropical coastal watershed of Southeast China. The landscape characteristics include Percent of Built (%BL), Percent of Agriculture, Percent of Natural, Patch Density and Shannon's Diversity Index (SHDI), with water quality expressed in terms of COD(Mn) and NH(4)(+)-N. The %BL was consistently positively correlated with NH(4)(+)-N and COD(Mn) at time three points. SHDI is significantly positively correlated with COD(Mn) in 2002. The relationship between NH(4)(+)-N, COD(Mn) and landscape variables in the wet precipitation year 2007 is stronger, with R(2) = 0.892, than that in the dry precipitation years 1996 and 2002, which had R(2) values of 0.712 and 0.455, respectively. Two empirical regression models constructed in this study proved more suitable for predicting COD(Mn) than for predicting NH(4)(+)-N concentration in the unmonitored watersheds that do not have wastewater treatment plants. The calibrated regression equations have a better predictive ability over space within the wet precipitation year of 2007 than over time during the dry precipitation years from 1996 to 2002. Results show clearly that climatic variability influences the linkage of water quality-landscape characteristics and the fit of empirical regression models.

Seasonal variation for the ratio of BaP to BeP at different sites in Great Xiamen Bay.

From March 2008 to February 2009, PM(10) samples were collected and analyzed for polycyclic aromatic hydrocarbons (PAHs) at eight sampling sites in Great Xiamen Bay, China. Analyses of the seasonal and spatial variations of these compounds revealed the following results. Significantly high levels of PAHs were found in the winter compared to the summer, sometimes exceeding 100 ng m(-3), and the spatial variations were influenced most by the sampling site surroundings. Composition profiles of PAHs of an urban and a rural site were shown to be very similar with a positive correlation coefficient larger than 0.9 at the 0.01 level of significance for the same season. Diagnostic ratios, together with principal component and multiple linear regression analysis, showed that more PAHs were from grass/wood/coal combustion in winter than in other seasons. The ratios of benzo[a]pyrene to benzo[e]pyrene (BaP-BeP) in winter and fall were 0.6-1.7 times higher than those in spring and summer, suggesting the importance of local emissions of PAHs. The BaP-BeP ratios in Kinmen were generally lower than those in Xiamen, indicating that the aging degree of PAHs was higher in Kinmen than in Xiamen. The external input of PAHs from upwind urban and industrial areas was one of the key factors causing high levels of PAHs in PM(10) in Great Xiamen Bay in winter.

Photo-production of dissolved inorganic carbon from dissolved organic matter in contrasting coastal waters in the southwestern Taiwan Strait, China.

Photo-production of dissolved inorganic carbon (DIC) from chromophoric dissolved organic matter (CDOM) is an important transformation process in marine carbon cycle, but little is known about this process in Chinese coastal systems. This study investigated an estuarine water sample and a coastal seawater sample from the subtropical waters in southeast of China. Water samples were exposed to natural sunlight and the absorption and fluorescence of CDOM as well as the DIC concentration were measured in the summer of 2009. The estuarine water had higher CDOM level, molecular weight and proportion of humic-like fluorescent components than the seawater that exhibited abundant tryptophan-like fluorescent component. After a 3-day irradiation, the CDOM level decreased by 45% in the estuarine water and 20% in the seawater, accompanied with a decrease in the molecular weight and aromaticity of DOM which was inferred from an increase in the absorption spectral slope parameter. The photo-degradation rates of all the five fluorescent components were also notable, in particular two humic-like components (C4 and C5) were removed by 78% and 69% in the estuarine water and by 69% and 56% in the seawater. The estuarine water had a higher photo-production rate of DIC than the seawater (4.4 vs. 2.5 micromol/(L x day)), in part due to its higher CDOM abundance. The differences in CDOM compositions between the two types of waters might be responsible for the higher susceptibility of the estuarine water to photo-degradation and hence could also affect the photo-production process of DIC.

Diurnal variation of nitrated polycyclic aromatic hydrocarbons in PM10 at a roadside site in Xiamen, China.

Intensive daytime and nighttime sampling was carried out from 23 Oct to 31 Dec 2008 to investigate the occurrence of nitrated polycyclic aromatic hydrocarbons (NPAHs) in PM10 at a roadside site in Xiamen, China. At the same time, six PM10 samples were collected from a nearby roadway tunnel for comparison. Six NPAHs, namely 9-nitroanthracene, 2- and 3-nitrofluoranthene, 1-nitropyrene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene, were identified and quantified using GC/MS in negative ion chemical ionization mode. The average total concentration of six NPAHs (sigmaNPAHs) in the cold season (26 Nov-31 Dec) was 2.3 (daytime) and 9.9 (nighttime) times higher than those in the warm season. Significant statistical difference (p < 0.01, 2-tailed) of sigmaNPAHs between daytime and nighttime was found during both the warm and cold seasons. NPAHs were significantly positively correlated with their parent PAHs and nitrogen dioxide but negatively correlated with ambient temperature. The ratio of 2 + 3-nitrofluoranthene to 1-nitropyrene exhibited a similar diurnal pattern as sigmaNPAHs and was generally greater than 5, indicating the importance of secondary atmospheric formation. The diurnal variations of NPAHs were all influenced by the diurnal variations of PAHs, nitrogen dioxide, sunlight, and temperature. The daily inhalable exposure to the six NPAHs in the tunnel was much higher than the roadside values in the warm season but only slightly higher than those in the cold season.

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry.

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions.

Homology-Driven Proteomics of Dinoflagellates with Unsequenced Genomes Using MALDI-TOF/TOF and Automated De Novo Sequencing.

This study developed a multilayered, gel-based, and underivatized strategy for de novo protein sequence analysis of unsequenced dinoflagellates using a MALDI-TOF/TOF mass spectrometer with the assistance of DeNovo Explorer software. MASCOT was applied as the first layer screen to identify either known or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed after searching against the NCBInr database, the remainder was searched against the dinoflagellate expressed sequence tag database. In the last layer, those borderline and nonconfident hits were further subjected to de novo interpretation using DeNovo Explorer software. The de novo sequences passing a reliability filter were subsequently submitted to nonredundant MS-BLAST search. Using this layer identification method, 216 protein spots representing 158 unique proteins out of 220 selected protein spots from Alexandrium tamarense, a dinoflagellate with unsequenced genome, were confidently or tentatively identified by database searching. These proteins were involved in various intracellular physiological activities. This study is the first effort to develop a completely automated approach to identify proteins from unsequenced dinoflagellate databases and establishes a preliminary protein database for various physiological studies of dinoflagellates in the future.

Atmospheric nitrogen deposition and its long-term dynamics in a southeast China coastal area.

Measurements were conducted during 2004-2005 and 2009-2010 to characterize atmospheric nitrogen (N) deposition to the Jiulong River Estuary - Xiamen Bay area in southeast China. Isotopic analysis and long-term data (1990-2009) for inorganic N extracted from the national acid deposition dataset were used to determine the dominant source of atmospheric nitrate and N component dynamics. The results showed that the mean dissolved total N concentration in rain water for the three coastal area sites was 2.71 ± 1.58 mg N L(-1) (n = 141) in 2004. The mean dissolved inorganic N at the Xiamen site was 1.62 ± 1.19 mg N L(-1) (n = 46) in 2004-2005 and 1.56 ± 1.39 mg N L(-1) (n = 36) in 2009-2010, although the difference is not significant, nitrate turnover dominates the N component in the latter period. Total deposition flux over Xiamen was 30 kg N ha(-1) yr(-1), of which dry and wet deposition contributed 16% and 84%, respectively. Nitrate in wet deposition with low isotopic value (between -3.05 and -7.48‰) was likely to have mostly originated from combustion NO(x) from vehicle exhausts. The inorganic N in acid deposition exhibited a significant increase (mainly for nitrate) since the mid-1990s, which is consistent with the increased gaseous concentrations of NO(x) and expanding number of automobiles in the coastal city (Xiamen). The time series of nitrate anions and ammonium cations as well as pH values during the period 1990-2009 reflected an increasing trend of N emission with potential implication for N-induced acidification.

[Parallel factor analysis for excitation emission matrix fluorescence spectroscopy of dissolved organic matter from a reservoir-type river].

The fluorescent components and their distribution and variation of dissolved organic matter (DOM) were examined using excitation emission matrix fluorescence spectroscopy-parallel factor analysis technique (EEMs-PARAFAC) for samples collected during June, 2008 from Minjiang River, a typical subtropical reservoir-type river ecosystem. Three separate fluorescent components were identified by PARAFAC, including two dominant humic-like components (C1: < 250, 325/424 nm; C2: 270, 395/482 nm) and one protein-like component (C3: < 250, 280/358 nm), of which humic-like components were dominant. Fluorescence analysis provided a 'fingerprint' technique to trace the mixing of DOM between three tributaries in the upstream of Minjiang River. The nearly constant concentration and composition in the main stream of Minjiang River reflected the effect of dam construction Correlation and principal component analysis (PCA) revealed that humic-like components were principally derived from flushing of watershed soils, while protein-like component was formed from in-situ production which could be used as a proxy of the concentration of total dissolved nitrogen (TDN). Multi-linear regression of fluorescent components C2 and C3 can be used to trace the variation of dissolved organic carbon (DOC) concentration. This study demonstrates that Minjiang is a typical subtropical reservoir-type river which still keeps relatively 'unpolluted' aquatic environment.

An improved ion-exchange/diffusion method for 15N isotope tracing analysis of nitrate in surface waters from watersheds.

An improved method, suitable for collecting nitrate from surface waters in the watershed for 15N isotope tracing analysis, was developed on the basis of the anion exchange coupled with diffusion through systematic simulation and comparison experiments. The results showed that the nitrate could be separated and enriched from the waters efficiently by using the improved method. Being simple and practical in operation principle and procedures, cost-economic, and highly efficient in nitrate separation/enrichment, the method met the requirements of delta(15)N mass spectrum analysis and would lay a foundation for the application of 15N isotope tracing approach to the research on non-point source pollution in watershed.

Hepcidin gene expression induced in the developmental stages of fish upon exposure to Benzo[a]pyrene (BaP).

Hepcidin is known to be expressed in fish with bacterial challenge and iron overload. Here we first report the hepcidin expression induced in the developmental stages from embryo to fry of red sea bream (Pagarus major) and in juvenile black porgy (Acanthopagrus schlegelii B.) upon continuous waterborne exposure to BaP. The gene expression of CYP1A1 and IgL (immunoglobulin light chain) were both measured. Expression of the Pagarus major hepcidin gene (PM-hepc) was increased in post hatch fry at 24 h and 120 h exposure to BaP at concentrations of 0.1, 0.5 and 1.0 microg/l, respectively. The gene expression pattern was comparable to that of CYP1A1 but different from that of IgL. In addition, a high number of AS-hepc2 transcripts (Acanthopagrus schlegelii B. hepcidin gene) were detected in the liver upon exposure to 1.0 microg/l BaP. This study demonstrates that hepcidin gene expression is significantly induced in BaP-exposed red sea bream and black porgy.

Seasonal variation and spatial transport of polycyclic aromatic hydrocarbons in water of the subtropical Jiulong River watershed and estuary, Southeast China.

Riverine runoff is one of the most important pathways of pollutants entering the oceans. To study the seasonal variations, spatial transports, sources and mass fluxes of polycyclic aromatic hydrocarbons (PAHs) from the subtropical Jiulong River watershed to estuary, water samples were collected in wet and dry seasons. PAH concentrations showed significant temporal-spatial variations (ANOVA, p < 0.05). In the watershed, PAH concentrations in wet season (48.6 ±â€¯18.2 ng L-1) were significantly lower than in dry season (90.3 ±â€¯18.5 ng L-1). In contrast, estuarine PAH concentrations in wet season (67.1 ±â€¯24.6 ng L-1) were significantly higher than in dry season (27.4 ±â€¯10.6 ng L-1) (p < 0.0001). The spatial variations of PAH concentrations in wet and dry seasons reflected positive and restricted transport processes occurred in the river. These findings might be subjected to seasonal changes in precipitation, water discharge, hydrodynamic conditions, and human activities. The compositional patterns of PAHs illustrated that fluorene and phenanthrene were the dominant compounds in the watershed, while phenanthrene was predominant in the estuary. Source analysis by molecular diagnostic ratios and PMF model indicated that fossil fuel and biomass combustion and petroleum both contributed to the presence of PAHs, and the high contributions of pyrogenic PAHs might be related to urban rainstorm runoff in winter and atmospheric inputs in winter. Although the estimated flux of PAHs from watershed to estuary was about 676 kg yr-1 with a low level by comparing the data obtained in the worldwide, continue concern of PAHs in the Jiulong River is recommended due to the intense human activities.

Environmental behavior of organotin compounds in the coastal environment of Xiamen, China.

In 2006, organotins pollution were investigated in the coastal environment of Xiamen, China. Six species of organotin compounds including tributyltin, triphenyltin and their degradation compounds were quantified in the dissolved and particulate phases of the water, and in the sediment using GC-FPD. The concentrations of organotin compounds ranged from 2.2 to 160 ng (Sn) L(-1) dissolved in the water, 0.14-6.7 ng (Sn) L(-1) in suspended particulate matter and nd approximately 26 ng (Sn) g(-1) (dry weight) in the sediment. The highest concentration of total organotin or tributyltin in water was found in a shipyard and at a station near the inlet of the harbor, indicating fresh inputs of antifouling paints to Xiamen's coastal environment. Organotin speciation was performed on sediment cores to investigate contamination trends over the past ten years in the harbor. The results of (210)Pb dating indicated that Xiamen western harbor suffered contamination during 2000. The environmental behavior of organotins such as the enhancement of the microlayer, partitioning between water/suspended particulate matter and between water/sediment are also discussed in this paper.

Validation of an agricultural non-point source (AGNPS) pollution model for a catchment in the Jiulong River watershed, China.

This study tested and evaluated the agricultural non-point source (AGNPS) model for the Wuchuan catchment, a typical agricultural area in the Jiulong River watershed, Fujian Province, China. The AGNPS model was calibrated and validated for the study area with observed data on ten storms. The data on eight storms in 2002 were used for calibration while data on two storms were used for validation of the model. Considering the lack of water quality data over a long-term series, a novel method, comparing an internal nested catchment with its surrounding catchment, was used to supplement the less long-term series data. Dual calibration and validation of the AGNPS model was obtained by this comparison. The results indicate that the correlation coefficients were 0.99 and 0.98 for runoff, 0.94 and 0.95 for the peak runoff rate of the large catchment and the small catchment, respectively, and 0.76 for the sediment of the small catchment only. Each pair of correlation coefficients is homogeneous for the same event for the two catchments. With the exception of the sediment yield and particulate phosphorus, the peak runoff rate and other nutrients were well predicted. Sensitivity analysis showed that the Soil Conservation Service curve number and rainfall quantity were the most sensitive parameters, which resulted in high output variations. Erosivity and other parameters had little influence on the hydrological and quality outputs.

Modeling nutrient sources, transport and management strategies in a coastal watershed, Southeast China.

Integrated watershed management requires an analytical model capable of revealing the full range of impacts that would be caused by the uses and developments in the watershed. The SPAtially Referenced Regressions On Watershed Attributes (SPARROW) model was developed in this study to provide empirical estimates of the sources, transport of total nitrogen (TN) and total phosphorus (TP) and to develop nutrient management strategies in the Jiulong River Watershed, southeast China that has enormous influence on the region's ecological safety. We calibrated the model using data related to daily streamflow, monthly TN and TP concentrations in 2014 at 30 locations. The model produced R2 values for TN with 0.95 and TP with 0.94. It was found that for the entire watershed, TN came from fertilizer application (43%), livestock breeding (39%) and sewage discharge (18%), while TP came from livestock breeding (46%), fertilizer application (46%), and industrial discharge (8%). Fifty-eight percent of the TN and 80% of the TP in upstream reaches are delivered to the outlets of North and West rivers. A scenario analysis with SPARROW was coupled to develop suitable management strategies. Results revealed that controlling nutrient sources was effective in improving water quality. Normally sharp reduction in nutrient sources is not operational feasible. Hence, it is recommended that preventing nutrient on land from entering into the river as a suitable strategy in watershed management.

Storm induced estuarine turbidity maxima and controls on nutrient fluxes across river-estuary-coast continuum.

Climate change is likely to increase the frequency and intensity of tropical storms. However, the impacts of major storms on nutrient cycling processes in the river-estuary-coast continuum are poorly understood. Continuous observations were made at lower river stations and on a transect down the Jiulong River Estuary in south east China for three storms in 2013-2014. There were major increases in both dissolved nutrients and suspended particulate matter (SPM) brought down the river during storms. Strong Estuarine Turbidity Maxima (ETM) were observed during major storms and were the result of SPM brought down rivers augmented by sediment scoured within the Upper Estuary (salinity=0psu) and possibly also from behind the dikes opened for flood control. There were major increases in dissolved nutrients (nitrate, ammonium and phosphate) in the Upper Estuary particularly during major Storms C (July 2013) and D (May 2014). These increases were probably due to river inflows with surrounding runoff, pore water supply and nutrients desorbed from scoured sediment. During major Storm D there were greater nutrient fluxes through the estuary-coast interface compared to the nutrients supplied through the river-estuary interface while the opposite pattern was observed during normal flow. The increased supply of ammonium and phosphate to the coastal region caused increased chlorophyll a once the light inhibiting SPM had been removed from the water column. This is likely to increase the potential of eutrophication. Storm induced increases in N:P:Si supplied from the estuary to the coastal region increased the degree of P limitation.

An efficient method to obtain axenic cultures of Alexandrium tamarense--a PSP-producing dinoflagellate.

The fact that species of harmful algae maintained in the laboratory harbor a complex bacterial flora increases the difficulties involved in the study of the relationship between bacteria and algae. An efficient method to remove bacteria from a laboratory culture of the marine dinoflagellate Alexandrium tamarense is presented in this paper. The alga was subjected to repeated washing, lysozyme/SDS and antibiotic treatment with a mixture of gentamycin, streptomycin, cephalothin and rifampicin. Axenic status was confirmed after subculturing three times in sterile f/2 medium without antibiotics. Bacteria could not be detected in various media, both solid and liquid, nor by epifluorescence microscopy and PCR amplification of 16S rDNA of both eubacteria and archaea. Bacterial presence was monitored throughout a full growth cycle and, following subculture, no bacteria were detected using the above methods. This method is more efficient and less time-consuming than other methods and the resultant axenic A. tamarense cultures would provide a simpler system for further study of bacteria-alga interactions.

Ecological anomalies in the East China Sea: impacts of the Three Gorges Dam?

In this study, we examined possible impacts of the Yangtze River Three Gorges Dam (TGD), the world largest hydroelectric construction, on the adjacent marine ecosystem of the East China Sea (ECS) during its initial water storage period. The TGD filled the first one-third of its storage capacity of 39 billion m3 in 10 days in June 2003, causing an abrupt reduction in the river flow into the ECS. Noticeable changes in the microbial community structure including pico-sized autotrophs, heterotrophic bacteria and microbial diversity in the estuary and the ECS were observed 2 months later. Although causes for these changes could be multiple, the sudden decrease of river runoff and an ensuing intrusion of ECS ocean currents were postulated to be among the major ones.