Taxonomic differences shape the responses of freshwater aerobic anoxygenic phototrophic bacterial communities to light and predation

Aerobic anoxygenic phototrophic (AAP) bacteria are a phylogenetically diverse and ubiquitous group of prokaryotes that use organic matter but can harvest light using bacteriochlorophyll a. Although the factors regulating AAP ecology have long been investigated through field surveys, the few available experimental studies have considered AAPs as a group, thus disregarding the potential differential responses between taxonomically distinct AAP assemblages. Here, we used sequencing of the pufM gene to describe the diversity of AAPs in 10 environmentally distinct temperate lakes, and to investigate the taxonomic responses of AAP communities in these lakes when subjected to similar experimental manipulations of light and predator removal. The studied communities were clearly dominated by Limnohabitans AAP but presented a clear taxonomic segregation between lakes presumably driven by local conditions, which was maintained after experimental manipulations. Predation reduction (but not light exposure) caused significant compositional shifts across most assemblages, but the magnitude of these changes could not be clearly related to changes in bulk AAP abundances or taxonomic richness of AAP assemblages during experiments. Only a few operational taxonomic units, which differed taxonomically between lakes, were found to respond positively during experimental treatments. Our results highlight that different freshwater AAP communities respond differently to similar control mechanisms, highlighting that in‐depth knowledge on AAP diversity is essential to understand the ecology and potential role of these photoheterotrophs.     

Diverse arrangement of photosynthetic gene clusters in aerobic anoxygenic phototrophic bacteria

Background

Aerobic anoxygenic photototrophic (AAP) bacteria represent an important group of marine microorganisms inhabiting the euphotic zone of the ocean. They harvest light using bacteriochlorophyll (BChl) a and are thought to be important players in carbon cycling in the ocean.

Methodology/Principal Findings

Aerobic anoxygenic phototrophic (AAP) bacteria represent an important part of marine microbial communities. Their photosynthetic apparatus is encoded by a number of genes organized in a so-called photosynthetic gene cluster (PGC). In this study, the organization of PGCs was analyzed in ten AAP species belonging to the orders Rhodobacterales, Sphingomonadales and the NOR5/OM60 clade. Sphingomonadales contained comparatively smaller PGCs with an approximately size of 39 kb whereas the average size of PGCs in Rhodobacterales and NOR5/OM60 clade was about 45 kb. The distribution of four arrangements, based on the permutation and combination of the two conserved regions bchFNBHLM-LhaA-puhABC and crtF-bchCXYZ, does not correspond to the phylogenetic affiliation of individual AAP bacterial species. While PGCs of all analyzed species contained the same set of genes for bacteriochlorophyll synthesis and assembly of photosynthetic centers, they differed largely in the carotenoid biosynthetic genes. Spheroidenone, spirilloxanthin, and zeaxanthin biosynthetic pathways were found in each clade respectively. All of the carotenoid biosynthetic genes were found in the PGCs of Rhodobacterales, however Sphingomonadales and NOR5/OM60 strains contained some of the carotenoid biosynthetic pathway genes outside of the PGC.

Conclusions/Significance

Our investigations shed light on the evolution and functional implications in PGCs of marine aerobic anoxygenic phototrophs, and support the notion that AAP are a heterogenous physiological group phylogenetically scattered among Proteobacteria.     

Abundance and diversity of aerobic anoxygenic phototrophic bacteria in saline lakes on the Tibetan plateau

Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic prokaryotes that are capable of utilizing light as an energy source but are not capable of producing molecular oxygen. Recently, multiple studies have found that AAP bacteria are widely distributed in oceans and estuaries and may play an important role in carbon cycling. However, AAP bacteria in inland lake ecosystems have not been investigated in depth. In this study, the abundance and diversity of the pufL-M genes, encoding photosynthetic reaction centers of AAP bacteria, were determined in the oxic water column and anoxic sediments of saline lakes (Qinghai, Erhai, and Gahai Lakes) on the Tibetan Plateau, China. Our results indicated that AAP bacteria were abundant in inland lakes, with the proportion of AAP bacteria (in total bacteria) comparable to those in the oceans, but with a lower diversity. Salinity and pH were found to be potential factors controlling the AAP bacterial diversity and community composition. Our data have implications for a better understanding of the potential role of AAP bacteria in carbon cycling in inland lake ecosystems.     

Diversity and distribution of ecotypes of the aerobic anoxygenic phototrophy gene pufM in the Delaware estuary

The diversity of aerobic anoxygenic phototrophic (AAP) bacteria has been examined in marine habitats, but the types of AAP bacteria in estuarine waters and distribution of ecotypes in any environment are not well known. The goal of this study was to determine the diversity of AAP bacteria in the Delaware estuary and to examine the distribution of select ecotypes using quantitative PCR (qPCR) assays for the pufM gene, which encodes a protein in the light reaction center of AAP bacteria. In PCR libraries from the Delaware River, pufM genes similar to those from Beta- (Rhodoferax-like) or Gammaproteobacteria comprised at least 50% of the clones, but the expressed pufM genes from the river were not dominated by these two groups in August 2002 (less than 31% of clones). In four transects, qPCR data indicated that the gammaproteobacterial type of pufM was abundant only near the mouth of the bay whereas Rhodoferax-like AAP bacteria were restricted to waters with a salinity of <5. In contrast, a Rhodobacter-like pufM gene was ubiquitous, but its distribution along the salinity gradient varied with the season. High fractions (12 to 24%) of all three pufM types were associated with particles. The data suggest that different groups of AAP bacteria are controlled by different environmental factors, which may explain current difficulties in predicting the distribution of total AAP bacteria in aquatic environments.     

玛珥湖好氧不产氧光合细菌pufM基因DNA和mRNA的定量及多样性分析

【目的】湖光岩玛珥湖是一类特殊的火山口湖,它完全封闭,地质年代久远,尚未受人类活动的剧烈影响,孕育着丰富而特殊的微生物种群。好氧不产氧光合细菌(AAPB)是以其在有氧情况下能行使光合功能而定义的一类专性异养细菌,其生理生态特征独特,进化年代久远,在水生生态系统的上层水体中广泛分布。目前,AAPB在玛珥湖水体中是否有分布仍是未知。【方法】构建和比较夏季湖水1 m、5 m、12 m三个水层的总DNA和总RNA的AAPB光合中心合成的关键基因pufM的6个克隆文库,并结合定量PCR技术,分析了不同水层AAPB的分布、系统发育多样性及其在总细菌中的比重。【结果】6个文库覆盖率和稀释曲线显示样本初步揭示了各水层优势AAPB类群的多样性。BLAST核苷酸同源性介于80%93%;多样性指数表明,湖光岩表层和底层多样性相当,中间层最低,总RNA的多样性高于总DNA。系统发育分析结果表明,OTU21 24所含的序列(占总序列的49.43%)与β-变形细菌的进化距离最接近,是湖光岩玛珥湖的优势AAPB菌群。定量PCR结果显示1 m水层中AAPB在总细菌中的比重最高,可达38.06%;而5 m水层中AAPB所占的比重最低,仅为0.85%;12 m为9.54%。【结论】湖光岩玛珥湖孕育着丰富而多样的AAPB类群。     

Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary

Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell ³H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware coast from August through December and on transects through the Delaware estuary in August and November 2011. The percent of active AAP bacteria was up to twofold higher than the percentage of active cells in the rest of the bacterial community in the estuary. Likewise, the silver grain area around active AAP bacteria in microautoradiography preparations was larger than the area around cells in the rest of the bacterial community, indicating higher rates of leucine consumption by AAP bacteria. The cell size of AAP bacteria was 50% bigger than the size of other bacteria, about the same difference on average as measured for activity. The abundance of AAP bacteria was negatively correlated and their activity positively correlated with light availability in the water column, although light did not affect ³H-leucine incorporation in light–dark experiments. Our results suggest that AAP bacteria are bigger and more active than other bacteria, and likely contribute more to organic carbon fluxes than indicated by their abundance.     

Dynamics of aerobic anoxygenic phototrophic bacteria in the East China Sea

Aerobic anoxygenic phototrophic bacteria (AAPB) are a group of heterotrophic bacteria capable of photosynthesis. The dynamics of AAPB in the East China Sea, a typical marginal sea characterized by diverse physical-chemical and ecological conditions, were investigated from April 2002 to September 2003. The results showed that the abundance of AAPB varied from 0.16 to 7.9 x 10(4) cells mL(-1) and the percentage of AAPB (AAPB%) in the total heterotrophic bacterial abundance varied from 0.5% to 11.6% over a gradient of environmental conditions. The abundance of AAPB and AAPB% was higher in coastal and continental shelf waters than in oceanic waters. An interesting seasonal pattern was observed in the Yangtze River estuary: the abundance of AAPB was highest in summer and lowest in winter; however, AAPB% was higher in winter than in the other seasons. Throughout the investigation period, variation of AAPB abundance with temperature was much less than that of nonAAPB abundance, suggesting that low temperature was not a limiting factor for AAPB in this case. Close correlation between AAPB and chlorophyll a was observed in each season, suggesting that dependence of AAPB on dissolved organic carbon produced by phytoplankton (PDOC) may be one key factor controlling AAPB distribution.     

Distribution of aerobic anoxygenic phototrophs in temperate freshwater systems

The presence of aerobic anoxygenic phototrophs (AAPs) was recently reported from various marine environments; however, there is little information regarding their distribution in fresh waters. We surveyed a number of freshwater systems in central Europe, by infra-red fluorometry, infra-red epifluorescence microscopy, fluorescence emission spectroscopy and pigment analyses. AAPs were found to be abundant in several oligotrophic and mesotrophic lakes (50–400 ng of bacteriochlorophyll a l⁻¹, 10–80% of bacterial biomass), while in more eutrophized water bodies they represented a negligible part of the total microbial community (< 1%). The observed freshwater AAPs were morphologically diverse and different from previously observed marine species. Under temperate European climatic conditions, AAP populations undergo strong seasonal changes in terms of both abundance and species composition, with the maximum biomass in summer and the minimum in winter. In the mountain lakes Čertovo and Plešné, AAPs contributed more than one half of total bacterial biomass during their summer maximum. These results show that photoheterotrophic bacteria represent an important part of the microbial community in many freshwater systems.     

High abundances of aerobic anoxygenic phototrophs in saline steppe lakes

We studied the distribution of anoxygenic phototrophs in 23 steppe lakes in the Transbaikal region (Russia), in Uzbekistan (Central Asia) and in the Crimean peninsula (Ukraine). The lakes varied in their mineral content and composition (salinities from 0.2 to 300 g L(-1) ). The Transbaikal lakes were alkaline (pH>9), with high amounts of soda. The Uzbek and Crimean lakes were more pH neutral, frequently with high amounts of sulfates. The presence of anoxygenic phototrophs was registered by infrared epifluorescence microscopy, infrared fluorometry and pigment analyses. In mostly shallow, fully oxic lakes, the anoxygenic phototrophs represented 7-65% of the total prokaryotes, with the maxima observed in Transbaikal soda lakes Gorbunka (32%), Khilganta (65%), Zanday (58%) and Zun-Kholvo (46%). Some of the lakes contained over 1 μg bacteriochlorophyll L(-1) . In contrast, only small amounts of anoxygenic phototrophs were present in highly mineralized lakes (>100 g total salts L(-1) ); Borzinskoe, Tsagan-Nur (Transbaikal), Staroe (Crimea) and in the residual part of the south-west Aral Sea (Uzbekistan). The oxic environment and the specific diurnal changes of bacteriochlorophyll concentration observed suggest that the phototrophic community was mostly composed of aerobic anoxygenic phototrophs. The high abundances and bacteriochlorophyll concentrations point to an important role of aerobic anoxygenic phototrophs in the habitats studied.     

The effect of the pH level on the communities of anoxygenic phototrophic bacteria of soda lakes of the southeastern Transbaikal region

The effect of pH on the structure of the communities of anoxygenic-phototrophic-bacteria (APB) was studied under laboratory conditions. Samples of natural APB communities were inoculated into media that differed in pH values, which were 7, 9.5, or 10.5. The structure of the APB communities in the obtained enrichment cultures depended on the pH values and on the mineralization levels of the media, which were the same as in the lakes from which samples were taken. The same dependence of the community structure on salinity was observed as in the case of the natural communities that had been described previously. APB were most diverse in the enrichment cultures grown at pH 9.5. The shift of the pH to either neutral or extremely alkaline values affected the species diversity within the APB community, resulting in marked predominance of the most adapted forms. It was shown that the status of Ectothiorhodospira within the community could serve as an indicator of both salinity and pH in soda lakes with a water mineralization of higher than 5 g/l. The statuses of various APB groups in the community as dependent on pH and salinity are discussed, as well as possible changes in these statuses due to changes in the water level and other environmental parameters in the studied lakes.     

Responses of aerobic anoxygenic phototrophic bacteria to algal blooms in the East China Sea

Aerobic anoxygenic phototrophic bacteria (AAPB) are a new functional group of heterotrophic bacteria capable of phototrophy, and are suggested to be closely related with phytoplankton. However, less known is the relationship between AAPB and dominant phytoplankton populations. In this tudy, the responses of AAPB to algal blooms (ABs) in the AB-frequent-occurrence area of the East China Sea were investigated during four cruises from March to June 2005, using an advanced ‘Time-series observation-based cyanobacteria-calibrated InfraRed Epifluorescence Microscopy (TIREM)’ approach. Generally, total bacterial abundances at the bloom stations were higher than or similar to those at the non-bloom stations during the same time period. Interestingly, the responses of AAPB to ABs seemed to be more diverse and complex. AAPB abundance was higher at the stations with ABs where Thalassiosira curviseriata Takano and Skeletonema costatum (Greville) Cleve, Noctiluca scintillans (Macartney) Kofoid et Swezy, or Prorocentrum donghaiense Lu and Karenia mikimotoi Hansen co-dominated than those at the non-bloom stations during the same time period. However at the stations with a bloom of Akashiwo sanguinea Hansen, AAPB abundance only accounted for ~20% of the average abundance of AAPB at the non-bloom stations. In addition, variations of AAPB’s proportion to total bacterial abundance (AAPB%) in response to ABs basically followed AAPB abundance. Overall, our results suggest that the responses of AAPB to ABs are AB-species specific and somewhat independent of chlorophyll a concentration.     

The effect of the pH level on the communities of anoxygenic phototrophic bacteria of soda lakes of the southeastern Transbaikal Region

The effect of pH on the structure of the communities of anoxygenic phototrophic bacteria (APB) was studied under laboratory conditions. Samples of natural APB communities were inoculated into media that differed in pH values, which were 7, 9.5, or 10.5. The structure of the APB communities in the obtained enrichment cultures at all pH values depended also on the mineralization levels of the media, which were the same as in the lakes from which samples were taken. The same dependence of the community structure on salinity was observed as in the case of the natural communities that had been described previously. APB were most diverse in the enrichment cultures grown at pH 9.5. The shift of the pH to either neutral or extremely alkaline values restricted the species diversity within the APB community, resulting in marked predominance of the most adapted forms. It was shown that the status of Ectothiorhodospira species within the community could serve not only as an indicator of salinity but also as an indicator of pH in soda lakes with a water mineralization of higher than 5 g/l. The statuses of various APB groups in the community as dependent on pH and salinity are discussed, as well as possible changes in these statuses due to changes in the water level and other environmental parameters in the studied lakes.     

Effects of gilvin on the composition and dynamics of metalimnetic communities of phototrophic bacteria in freshwater North-American lakes

The spectral distribution of light reaching the populations of phototrophic bacteria in the metalimnion of stratified lakes is a selective factor determining the community composition. At deep metalimnia, light spectra are enriched in photons of the central part of the spectrum (500-600 nm) and benefit Chromatiaceae, brown-coloured Chlorobiaceae and phyco-erythrine-containing cyanobacteria. Their carotenoids (okenone, spiriloxanthine, isorenieratene) and phycoerythrines allow these phototrophic bacteria to use light from the narrow central spectral wavebands. Otherwise, shallow metalimnetic communities receive light from a wide range (400-800 nm) and their composition is more diverse and usually enriched in green-coloured Chlorobiaceae, which are unable to take advantage of the central part of the spectrum. Gilvin compounds (humic substances dissolved in water), have strong effects on light absorption, especially at shorter wavelengths. Therefore, light spectra in lakes with high gilvin contents are enriched in photons of long wavelengths (> 600 nm). Several Wisconsin lakes with different gilvin contents were studied during the period of summer stratification in 1994. Spectral distribution of light reaching their metalimnia changed with increasing gilvin contents (measured as g(440) ). In the latter, phototrophic metalimnetic bacterial communities were absolutely dominated by green-coloured Chlorobiaceae. Intermediate lakes could experiment changes on their community composition depending on variations in gilvin content, as happened in Little Long lake. The dynamics of this lake was studied during summer 1995. The ratio of green-coloured species in respect to brown-coloured species increased after a sudden increase of gilvin due to strong rainfall. These results agree with the photosynthetic advantage of green-coloured Chlorobiaceae under red-light illumination, inferred from laboratory experiments, and suggest a bacteriochlorophyll-dependent, light-harvesting strategy of these phototrophic sulphur bacteria.     

Real-time PCR for quantification of aerobic anoxygenic phototrophic bacteria based on pufM gene in marine environment

The abundance of aerobic anoxygenic phototrophic bacteria (AAPB), a new functional group that plays important roles in marine carbon cycling, is determined frequently by infrared epifluorescence microscopic analysis (IREM) or high-performance liquid chromatography (HPLC) based on detecting BChl a (bacteriochlorophyll a) fluorescence signal at 880 nm. Unfortunately, the fluorescence signal is often influenced by environmental variables and physiological state of cell. Here we developed a real-time quantitative PCR (qPCR) assay based on pufM gene to specifically quantify AAPB in marine environments. High specificity and sensitivity for estimation of AAPB abundance were revealed by analysis of amplification products, melting curves and target sequences. The phylogenetic tree indicated that this primer set is suitable for a wide genetic diversity of AAPB, including α-3, α-4 Proteobacteria and clones of unclear taxonomic position. In contrast, no amplicon was obtained from green non-sulphur bacteria and oxygenic phototrophic bacteria such as Cyanobacterial genomic DNA. The melting behavior could indicate predominant phenotypes in AAPB community in addition to validating the products of qPCR. The AAPB was estimated to range from 1.3 × 10⁴ cell/ml to 3.4 × 10⁵ cell/ml in our 10 tested water samples by this qPCR assay. Further investigations on the abundance distribution of AAPB in marine environments using the qPCR assay may provide new insight into their ecological functions.     

Distinct distribution pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global ocean

Aerobic anoxygenic phototrophic bacteria (AAPB) are an important bacterial group with capability of harvesting light energy, and appear to have a particular role in the ocean's carbon cycling. Yet the significance of AAPB relative to total bacteria (AAPB%) in different marine regimes are still controversial, and variation trend of genetic diversity of AAPB along environmental gradients remains unclear. Here we present the first comprehensive observation of the global distribution of AAPB in the Pacific, Atlantic and Indian oceans, revealing a general pattern of high abundance of AAPB and AAPB% in coastal waters than oceanic waters. The Indian Ocean contained relatively high AAPB% compared with the other two oceans, corresponding to the high primary production in this region. Both abundance of AAPB and AAPB% were positively correlated with the concentration of chlorophyll a, while the diversity of AAPB decreased with increasing chlorophyll a values. Our results suggest that AAPB abundance and diversity follow opposite trends from oligotrophic to eutrophic regimes in the ocean.     

Microbial ecology of planktonic filamentous phototrophic bacteria in holomictic freshwater lakes

The microbial ecology of the filamentous phototrophic bacterium, Chloronema giganteum, has been studied in the water column of three central European lakes (Schlein, Buchen and Vechten). In these lakes an anoxic layer, termed the transition zone, was located between the oxycline and the redoxcline. The migration capacity of Chloronema through this zone appears to be responsible for the natural preponderance of either straight or spiral forms. When the transition zone is less than 1 m thick the straight form is dominant, but when this transition zone is wider than 2 m the spiral form is enriched. The intermediate situations favour both filamentous forms.     

Abundance and genetic diversity of aerobic anoxygenic phototrophic bacteria of coastal regions of the pacific ocean

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophic microbes that are found in a broad range of aquatic environments. Although potentially significant to the microbial ecology and biogeochemistry of marine ecosystems, their abundance and genetic diversity and the environmental variables that regulate these properties are poorly understood. Using samples along nearshore/offshore transects from five disparate islands in the Pacific Ocean (Oahu, Molokai, Futuna, Aniwa, and Lord Howe) and off California, we show that AAP bacteria, as quantified by the pufM gene biomarker, are most abundant near shore and in areas with high chlorophyll or Synechococcus abundance. These AAP bacterial populations are genetically diverse, with most members belonging to the alpha- or gammaproteobacterial groups and with subclades that are associated with specific environmental variables. The genetic diversity of AAP bacteria is structured along the nearshore/offshore transects in relation to environmental variables, and uncultured pufM gene libraries suggest that nearshore communities are distinct from those offshore. AAP bacterial communities are also genetically distinct between islands, such that the stations that are most distantly separated are the most genetically distinct. Together, these results demonstrate that environmental variables regulate both the abundance and diversity of AAP bacteria but that endemism may also be a contributing factor in structuring these communities.     

Communities of anoxygenic phototrophic bacteria in the saline soda lakes of the Kulunda steppe (Altai Krai)

The saline soda lakes of the Kulunda steppe (Altai krai) are small and shallow; they are characterized by a wide range of salinity and alkalinity, as well as by the extreme instability of their water and chemical regimes. Accumulations of anoxygenic phototrophic bacteria (APB) visible to the unaided eye were noted only in several lakes with high rates of sulfate reduction in their bottom sediments. However, enumeration of APB cells by inoculation revealed their presence in all 17 lakes. APB cell numbers varied from 10³ to 10⁹ CFU cm^?3. In the APB communities of all lakes, purple sulfur bacteria of the family Ectothiorhodospi- raceae were predominant. In 14 out of the 17 lakes, purple nonsulfur bacteria of the family Rhodobacteraceae were also detected (10³–10⁷ CFU cm^?3). Purple sulfur bacteria of the family Chromatiaceae were less abundant: Halochromatium sp. (10⁴–10⁷ CFU cm^?3) were found in six lakes, while Thiocapsa sp. (10⁴ CFU cm³) were detected in one lake. On the whole, the APB communities of the soda lakes of the Kulunda steppe were characterized by the low diversity and evenness of their species compositions, as well as by the pronounced dominance of the members of the family Ectothiorhodospiraceae. There was no correlation between the structures of the APB communities and alkalinity. However, the dependence of the species composition of APB (mainly ectothiorhodospiras) on water mineralization was revealed. High mineralization (above 200 g l^?1) was a limiting factor that affected the APB communities on the whole, restricting the APB species diversity to extremely halophilic bacteria of the genus Halorhodospira.     

Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the Mediterranean Sea in relation to environmental variables

Aerobic anoxygenic phototrophic bacteria (AAP) represent an important fraction of bacterioplankton assemblages in various oceanic regimes. Although their abundance and distribution have been explored recently in diverse oceanic regions, the environmental factors controlling the population structure and diversity of these photoheterotrophic bacteria remain poorly understood. Here, we investigate the horizontal and vertical distributions and the genetic diversity of AAP populations collected in late summer throughout the Mediterranean Sea using pufM-temporal temperature gel gradient electrophoresis (TTGE) and clone library analyses. The TTGE profiles and clone libraries analyzed using multivariate statistical methods demonstrated a horizontal and vertical zonation of AAP assemblages. Physicochemical parameters such as pH, inorganic nitrogen compounds, photosynthetically active radiation, total organic carbon and to a lesser extent particulate organic nitrogen and phosphorus, and biogenic activities (e.g. bacterial production, cell densities), acted in synergy to explain the population changes with depth. About half of the pufM sequences were <94% identical to known sequences. The AAP populations were predominantly (~80%) composed of Gammaproteobacteria, unlike most previously explored marine systems. Our results suggest that genetically distinct ecotypes inhabiting different niches may exist in natural AAP populations of the Mediterranean Sea whose genetic diversity is typical of oligotrophic environments.