Bdellovibrio bacteriovorus gen. et sp. n., a predatory,ectoparasitic, and bacteriolytic microorganism

Bdellovibrio bacteriovorus, gen. et sp. n., a predatory and ectoparasitic microorganism with lytic activity against susceptible bacteria, is described, as are techniques for isolation and cultivation. These unusual bacteria cause reactions that are similar in their outward manifestations to bacteriophage-induced lysis. Upon plating a mixture of host bacteria and parasites, confluent lysis or single plaque formation occurs, just as in titration experiments with bacteriophage. However, the parasite plaques develop more slowly than phage plaques. Lysis of host bacteria in liquid culture is accompanied by a decrease in optical density; actually, a population of infected host bacteria is replaced by a population of the tiny parasite.Individual cells of the presently known strains ofBdellovibrio bacteriovorus are typically about 0.3 µ in width and, thus, are considerably narrower than ordinary bacteria. Therefore, they can pass Millipore filters of 0.45 µ pore size diameter. Their shape is often vibrio-like. They possess one unusually thick polar flagellum of about 50 mµ diameter, and they show a distinctive type of motility.The interaction betweenBdellovibrio and the attacked host bacterium can be followed in the phase-contrast microscope; it is characterized by a physical attack of the highly motile parasite, attachment to the bacterial cell surface, and lysis of the host cell.It has not yet been possible to cultivateBdellovibrio in its parasitic form on any artificial substrate. All parasitic strains require living host cells for their propagation. However, saprophytic mutants can be selected from a population of the parasite. These saprophytic derivatives are unable to lyse living bacteria as does the wild-type parasite. On the basis of morphological and physiological properties, a saprophyte strain which has been examined in some detail shows no close relationship to any of the already known categories of bacteria.A study of the kinetics of growth ofBdellovibrio in mixed culture with a susceptible host has disclosed that the number of parasites produced is not proportional to the number of host bacteria killed during the same period. After the majority of the host cells has been destroyed, there is still a considerable increase in parasites, indicating that they grow at the expense of material released from the lysed bacteria. Under the conditions of this trial, the generation time is about 100 minutes.All presently known isolates ofBdellovibrio possess lytic activity only against gram-negative bacteria. The individual strains, however, show certain differences in their host activity spectra; some have a restricted host range, while others are able to attack a broad spectrum of host bacteria.     

Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences

The significance of bacteria for eukaryotic functioning is increasingly recognized. Coral reef ecosystems critically rely on the relationship between coral hosts and their intracellular photosynthetic dinoflagellates, but the role of the associated bacteria remains largely theoretical. Here, we set out to relate coral‐associated bacterial communities of the fungid host species Ctenactis echinata to environmental settings (geographic location, substrate cover, summer/winter, nutrient and suspended matter concentrations) and coral host abundance. We show that bacterial diversity of C. echinata aligns with ecological differences between sites and that coral colonies sampled at the species’ preferred habitats are primarily structured by one bacterial taxon (genus Endozoicomonas) representing more than 60% of all bacteria. In contrast, host microbiomes from lower populated coral habitats are less structured and more diverse. Our study demonstrates that the content and structure of the coral microbiome aligns with environmental differences and denotes habitat adequacy. Availability of a range of coral host habitats might be important for the conservation of distinct microbiome structures and diversity.     

Prediction of surface exposed proteins in Streptococcus pyogenes,with a potential application to other Gram‐positive bacteria

The in silico prediction of bacterial surface exposed proteins is of growing interest for the rational development of vaccines and in the study of bacteria–host relationships, whether pathogenic or host beneficial. This interest is driven by the increase in the use of DNA sequencing as a major tool in the early characterization of pathogenic bacteria and, more recently, even of complex ecosystems at the host–environment interface in metagenomics approaches. Current protein localization protocols are not suited to this prediction task as they ignore the potential surface exposition of many membrane‐associated proteins. Therefore, we developed a new flow scheme, SurfG+, for the processing of protein sequence data with the particular aim of identification of potentially surface exposed (PSE) proteins from Gram‐positive bacteria, which was validated for Streptococcus pyogenes. The results of an exploratory case study on closely related lactobacilli of the acidophilus group suggest that the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) dedicates a relatively important fraction of its coding capacity to secreted proteins, while the probiotic gastrointestinal (GI) tract bacteria L. johnsonii and L. gasseri appear to encode a larger variety of PSE proteins, that may play a role in the interaction with the host.     

A freshwater radiation of diplonemids

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Japanese deep freshwater lakes. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and catalysed reporter deposition-fluorescent in situ hybridization (CARD-FISH). We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to a marine clade, which are mainly isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single-cell genomes of marine diplonemids show they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."     

Putting floodplain hyperdiversity in a regional context: an assessment of terrestrial–floodplain connectivity in a montane environment

Abstract Aim and location Alluvial flood plains support higher levels of vascular plant species richness than other terrestrial ecosystems. Whereas the spatial and temporal heterogeneity of these ecosystems has been considered the local determinant of high plant richness, regional influences, such as regional species pools have received little attention. In this study we surveyed plant species richness across the entire Nyack catchment (c. 21,000 ha), in Glacier National Park, USA, to determine the relation of upland ecosystem community structure to biodiversity patterns on montane floodplains that are relatively extensive and flood‐scoured ecosystems. Method We surveyed floodplain and other terrestrial ecosystems within the Nyack catchment using 50 × 2 m plots to record species present and visual estimates of percentage cover. Species pools from flood plains and three other terrestrial ecosystems (low elevation forests, sub‐Alpine forests and alpine) were analysed with nested subset analysis, detrended correspondence analysis (DCA), and an index of beta diversity to identify dissimilarity in species composition and richness, and the separate contributions of generalists (species occurring in more than one ecosystem) and specialists to richness in each ecosystem. Analysis of variance and post hoc Tukey–Kramer tests were used to identify where in the Nyack catchment each species was most abundant. Species life form and dispersal strategies were analysed to better understand influences on beta diversity. Results Our data show that in this pristine system, floodplain ecosystems host 202 (63%) of the 320 vascular plants identified within Nyack catchment. Of these species, the nested subset analysis showed that 146 (72%) are found in at least one adjacent upland ecosystem. While the DCA ordination scatter plots show statistically significant separations of ecosystems on the first two axes, values of beta diversity showed that substantial similarity exists between floodplain and all upland species pools. Further, of the 146 floodplain species shared with upland ecosystems, 61% were more frequent in upland ecosystems, whereas 55% were more abundant in uplands than flood plains (Tukey–Kramer P ≤ 0.05). Significant numbers of specialists were found on flood plains (24% of floodplain species), but also within upland ecosystems, where 23% and 40% of low elevation forest and alpine species were found to be specialists, respectively. Whereas 83% of herb generalists were wind dispersed, <70% of specialists were animal dispersed, indicating that similarity in species pools may be driven by wind dispersal. Main conclusions These results suggest a re‐evaluation of the contribution of floodplain ecosystems to regional plant species richness. While flood plains host specialists, other ecosystems had equal or higher levels of regional ‘endemism’. Furthermore, these data suggest that conservation of high levels of biodiversity on floodplain ecosystems may require consideration of upland ecosystems throughout the catchment as the majority of species were relatively rare on flood plains, indicating they may be sink habitats for some species.     

裂解或溶源-细菌遭遇噬菌体后的命运抉择

噬菌体是地球上数量最丰富的有机体,其在自然生态系统的塑造和细菌进化驱动中发挥着至关重要的作用。在与宿主的相互斗争中,噬菌体可以选择以下2种方式决定其与宿主的命运:(1)裂解:通过裂解宿主细胞最终大量释放噬菌体颗粒;(2)溶源:将其染色体整合到宿主细胞基因组中,与宿主建立一种潜在的互存关系。对于一些温和的噬菌体,这种倾向进一步受到感染多样性的调节,其中单一感染主要是裂解性的,而多重感染则多是溶源性的。溶源性的噬菌体不仅可以根据外界环境的理化因子,还可以通过细菌自身的群体感应系统来启动裂解-溶源开关,进而决定其宿主菌的命运。与此同时,宿主细菌在与噬菌体长时间的斗争中也进化出了针对噬菌体的手段。总而言之,噬菌体深刻影响着细菌的群落动态、基因组进化和生态系统等,而这一切都取决于噬菌体与宿主间的斗争模式(裂解/溶源性感染)。本文探讨了导致温和噬菌体对宿主菌进行裂解-溶源命运抉择的影响因素并系统性总结了细菌在面对噬菌体侵染时的应对策略的最新研究进展,以期能为噬菌体与宿主的研究提供建议和帮助。     

Construction of genetically engineered Streptococcus gordonii strains to provide control in QPCR assays for assessing microbiological quality of recreational water

Aims: Quantitative polymerase chain reaction (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for both amplification and cell lysis in assays measuring abundance of vegetative Gram‐positive bacteria. Methods and Results: Controls were constructed using Streptococcus gordonii DL‐1, a naturally transformable, Gram‐positive bacterium. Unique target sequences were provided by chromosomal insertion of a genetically modified, green fluorescent protein gene fragment. Results suggest that their use for control of lysis and amplification may be of significant value. Conclusions: The use of these controls and the establishment of data quality objectives to determine the tolerable level of decision error should ensure that environmental decisions based on QPCR data are technically and scientifically sound. Significance and Impact of the Study: QPCR measurements related to cell abundance may vary between samples as thick‐walled Gram‐positive bacteria are inherently difficult to lyse and substances present in recreational waters may inhibit amplification. As QPCR methods are considered for beach monitoring, it is essential to demonstrate that the data obtained accurately reflects the abundance of the bacterial indicator.     

Ecological generalism facilitates the evolution of sociality in snapping shrimps

Evidence from insects and vertebrates suggests that cooperation may have enabled species to expand their niches, becoming ecological generalists and dominating the ecosystems in which they occur. Consistent with this idea, eusocial species of sponge‐dwelling Synalpheus shrimps from Belize are ecological generalists with a broader host breadth and higher abundance than non‐eusocial species. We evaluate whether sociality promotes ecological generalism (social conquest hypothesis) or whether ecological generalism facilitates the transition to sociality (social transition hypothesis) in 38 Synalpheus shrimp species. We find that sociality evolves primarily from host generalists, and almost exclusively so for transitions to eusociality. Additionally, sponge volume is more important for explaining social transitions towards communal breeding than to eusociality, suggesting that different ecological factors may influence the independent evolutionary origins of sociality in Synalpheus shrimps. Ultimately, our results are consistent with the social transition hypothesis and the idea that ecological generalism facilitates the transition to sociality.     

Decoupled responses of soil bacteria and their invertebrate consumer to warming,but not freeze–thaw cycles,in the Antarctic Dry Valleys

Altered temperature profiles resulting in increased warming and freeze–thaw cycle (FTC) frequency pose great ecological challenges to organisms in alpine and polar ecosystems. We performed a laboratory microcosm experiment to investigate how temperature variability affects soil bacterial cell numbers, and abundance and traits of soil microfauna (the microbivorous nematode Scottnema lindsayae) from McMurdo Dry Valleys, Antarctica. FTCs and constant freezing shifted nematode body size distribution towards large individuals, driven by higher mortality among smaller individuals. FTCs reduced both bacterial and nematode abundance, but bacterial cell numbers also declined under warming, demonstrating decoupled consumer–prey responses. We predict that higher occurrence of FTCs in cold ecosystems will select for large body size within soil microinvertebrates and overall reduce their abundance. In contrast, warm temperatures without FTCs could lead to divergent responses in soil bacteria and their microinvertebrate consumers, potentially affecting energy and nutrient transfer rates in soil food webs of cold ecosystems.     

Non‐native species have multiple abundance–impact curves

The abundance–impact curve is helpful for understanding and managing the impacts of non‐native species. Abundance–impact curves can have a wide range of shapes (e.g., linear, threshold, sigmoid), each with its own implications for scientific understanding and management. Sometimes, the abundance–impact curve has been viewed as a property of the species, with a single curve for a species. I argue that the abundance–impact curve is determined jointly by a non‐native species and the ecosystem it invades, so that a species may have multiple abundance–impact curves. Models of the impacts of the invasive mussel Dreissena show how a single species can have multiple, noninterchangeable abundance–impact curves. To the extent that ecosystem characteristics determine the abundance–impact curve, abundance–impact curves based on horizontal designs (space‐for‐time substitution) may be misleading and should be used with great caution, it at all. It is important for scientists and managers to correctly specify the abundance–impact curve when considering the impacts of non‐native species. Diverting attention from the invading species to the invaded ecosystem, and especially to the interaction between species and ecosystem, could improve our understanding of how non‐native species affect ecosystems and reduce uncertainty around the effects of management of populations of non‐native species.     

It’s a wormy world: Meta‐analysis reveals several decades of change in the global abundance of the parasitic nematodes Anisakis spp. and Pseudoterranova spp. in marine fishes and invertebrates

The Anthropocene has brought substantial change to ocean ecosystems, but whether this age will bring more or less marine disease is unknown. In recent years, the accelerating tempo of epizootic and zoonotic disease events has made it seem as if disease is on the rise. Is this apparent increase in disease due to increased observation and sampling effort, or to an actual rise in the abundance of parasites and pathogens? We examined the literature to track long‐term change in the abundance of two parasitic nematode genera with zoonotic potential: Anisakis spp. and Pseudoterranova spp. These anisakid nematodes cause the disease anisakidosis and are transmitted to humans in undercooked and raw marine seafood. A total of 123 papers published between 1967 and 2017 met our criteria for inclusion, from which we extracted 755 host–parasite–location–year combinations. Of these, 69.7% concerned Anisakis spp. and 30.3% focused on Pseudoterranova spp. Meta‐regression revealed an increase in Anisakis spp. abundance (average number of worms/fish) over a 53 year period from 1962 to 2015 and no significant change in Pseudoterranova spp. abundance over a 37 year period from 1978 to 2015. Standardizing changes to the period of 1978–2015, so that results are comparable between genera, we detected a significant 283‐fold increase in Anisakis spp. abundance and no change in the abundance of Pseudoterranova spp. This increase in Anisakis spp. abundance may have implications for human health, marine mammal health, and fisheries profitability.     

Microorganisms Associated with Feathers of Barn Swallows in Radioactively Contaminated Areas Around Chernobyl

The Chernobyl catastrophe provides a rare opportunity to study the ecological and evolutionary consequences of low-level, environmental radiation on living organisms. Despite some recent studies about negative effects of environmental radiation on macroorganisms, there is little knowledge about the effect of radioactive contamination on diversity and abundance of microorganisms. We examined abundance patterns of total cultivable bacteria and fungi and the abundance of feather-degrading bacterial subset present on feathers of barn swallows (Hirundo rustica), a colonial migratory passerine, around Chernobyl in relation to levels of ground level environmental radiation. After controlling for confounding variables, total cultivable bacterial loads were negatively correlated with environmental radioactivity, whereas abundance of fungi and feather-degrading bacteria was not significantly related to contamination levels. Abundance of both total and feather-degrading bacteria increased with barn swallow colony size, showing a potential cost of sociality. Males had lower abundance of feather-degrading bacteria than females. Our results show the detrimental effects of low-level environmental radiation on total cultivable bacterial assemblage on feathers, while the abundance of other microorganism groups living on barn swallow feathers, such as feather-degrading bacteria, are shaped by other factors like host sociality or host sex. These data lead us to conclude that the ecological effects of Chernobyl may be more general than previously assumed and may have long-term implications for host–microbe interactions and overall ecosystem functioning.     

Niche‐based host extinction increases prevalence of an environmentally acquired pathogen

Understanding the ecology of environmentally acquired and multi‐host pathogens affecting humans and wildlife has been elusive in part because fluctuations in the abundance of host and pathogen species may feed back onto pathogen transmission. Complexity of pathogen‐host dynamics emerges from processes driving local extinction of the pathogen, its hosts and non‐hosts. While the extinction of species may entail losses in pathogen–host interactions and decrease the proportion of hosts infected by a pathogen (prevalence), some studies suggest the opposite pattern. Niche‐based extinction, based on the species tolerance to environmental conditions, may increase prevalence of infection because the pathogen and its hosts persist, while other species go extinct. Hence, understanding prevalence of infection requires disentangling random‐ and niche‐based extinction processes occurring simultaneously. To contribute to this exercise, we analysed the prevalence of an environmentally acquired, multi‐host pathogen, Mycobacterium ulcerans (MU), in a unique dataset of 16 communities of freshwater animals, surveyed during 12 months in Akonolinga, Cameroon in equatorial Africa. Two different ecosystems were identified: rivers (lotic) and swamps and flooded areas (lentic). Increased prevalence of MU infection was correlated with niche‐based extinction of aquatic host invertebrates and vertebrates in the lentic ecosystems, whereas decreased prevalence was associated with random disassembly of the lotic ecosystems. This finding suggests that random and niche‐based extinction of host taxa are key to assessing the effect of local extinction of species on the ecology of environmentally acquired and multi‐host pathogens.     

Altitudinal Distribution of Ammonia-Oxidizing Archaea and Bacteria in Alpine Grassland Soils Along the South-Facing Slope of Nyqentangula Mountains,Central Tibetan Plateau

Nitrogen is a major limiting nutrient for the net primary production of terrestrial ecosystems, especially on sentinel alpine ecosystem. Ammonia oxidation is the first and rate-limiting step on nitrification process and is thus crucial to nitrogen cycle. To decipher climatic influence on ammonia oxidizers, their communities were characterized by qPCR and clone sequencing by targeting amoA genes (encoding the alpha subunit of ammonia mono-oxygenase) in soils from 7 sites over an 800 m elevation transect (4400–5200 m a.s.l.), based on “space-to-time substitution” strategy, on a steppe-meadow ecosystem located on the central Tibetan Plateau (TP). Archaeal amoA abundance outnumbered bacterial amoA abundance at lower altitude (<4800 m a.s.l.), but bacterial amoA abundance was greater in surface soils at higher altitude (≥4800 m a.s.l.). Archaeal amoA abundance decreased with altitude in surface soil, while its abundance stayed relatively stable and was mostly greater than bacterial amoA abundance in subsurface soils. Conversely, bacterial amoA abundance gradually increased with altitude at all three soil depths. Statistical analysis indicated that altitude-dependent factors, in particular pH and precipitation, had a profound effect on the abundance and community of ammonia-oxidizing bacteria, but only on the community composition of ammonia-oxidizing archaea along the altitudinal gradient. These findings imply that the shifts in the relative abundance and/or community structure of ammonia-oxidizing bacteria and archaea may result from the precipitation variation along the altitudinal gradient. Thus, we speculate that altitude-related factors (mainly precipitation variation combing changed pH), would play a vital role in affecting nitrification process on this alpine grassland ecosystem located at semi-arid area on TP.     

Lysis of a red-tide causing alga,Alexandrium tamarense,caused by bacteria from its phycosphere

There are bacteria coexisting in xenic cultures of Alexandrium tamarense, a red-tide causing alga. However little is known concerning the interactions between the alga and the bacteria in its phycosphere. The objective of the current study was to determine the effect of the bacteria in its phycosphere on the growth of the alga. We added one percent (v/v) Zobell 2216E medium to A. tamarense culture to alter bacterial growth and the results showed that algal cells were all lysed within 14 h. After adding the medium, both the abundance and the extracellular enzyme activity of the bacteria increased by 50–100 times from the 4th to the 10th hour which resulted in lysis of the algae. The 16S rRNA gene fragments of the bacteria were amplified from the DNA extracted from A. tamarense cultures and analyzed by denaturing gradient gel electrophoresis and sequencing. The structure of the bacterial community in phycosphere changed significantly during algal lysis. Two bacterial genera, Alteromonas sp. and Thalassobius aestuarii sp. are key factors that caused the lysis, and the β-glucosidase and chitinase produced by the bacteria in the phycosphere could directly cause the lysis. These experiments provide evidence that bacteria in its phycosphere play a key role in the culture of A. tamarense, and may provide insights into the future biocontrol of red-tides.     

Single‐cell sequencing of dinoflagellate (Dinophyceae) nuclear ribosomal genes

Genetic sequences from dinoflagellates offer valuable information regarding taxonomies, phylogenies and population genetics that generally require the growth of these organisms in culture. We have developed a quick and simple method to obtain small and large subunit ribosomal gene sequences from dinoflagellates using single cells. This method, based on freeze–thaw cell lysis and a simple two‐step polymerase chain reaction, provides template for sequencing in 6–8 h. We have sequenced five dinoflagellate species, including unculturable Dinophysis and Ceratium species, using fresh and frozen samples.