Flood effects on invertebrates, sediments and particulate organic matter in the hyporheic zone of a gravel-bed stream

1. We investigated the effects of a flood on the fauna and physical habitat of the hyporheic zone of the Kye Burn, a fourth order gravel‐bed stream in New Zealand. 2. Freeze core hyporheic samples (to 50 cm depth) and benthic samples (to 10 cm) were taken, along with measurements of vertical hydrological gradient, before, 2 days after and 1 month after the flood (estimated return period: 1.5 years, estimated Q_max = 10.4 m³ s^?1). 3. The composition of the hyporheos differed over the three sampling occasions with fewer taxa collected immediately postflood than preflood. The equitability of the community was higher on both postflood occasions, consistent with the reduced densities of two abundant taxa (Leptophlebiidae and Copepoda). 4. Total invertebrate abundance was lower on the postflood occasions than preflood in both benthic (0–10 cm) and hyporheic (10–50 cm) sediments. Several taxa, including asellotan isopods and amphipods, recovered within 1 month of the event. Hyporheic densities of larval Hydora and nematodes did not differ among the three sampling occasions, but the water mite Pseudotryssaturus was more abundant 1 month after the flood than preflood. There was no evidence of vertical movements (to 50 cm) by any taxa in response to the flood. 5. The proportion of fine sediments (<1 mm) in the subsurface sediments (10–50 cm) increased over the three sampling occasions and median particle size declined, but sediment porosity did not change. More particulate organic matter was found in the sediments after the flood. 6. Our study provides little evidence that the hyporheic zone (to 50 cm) acted as a significant refuge during the flood event, although movements to or recolonisation from sediments deeper than 50 cm could explain the recovery of many crustacean and mite taxa within 1 month.     

Top‐down effects on pelagic heterotrophic nanoflagellates (HNF) in a large river (River Rhine): do losses to the benthos play a role?

1. The abundance, production and control of pelagic heterotrophic nanoflagellates (HNF) in the Lower River Rhine (Germany) were investigated. Field samples (live‐counting technique) were taken at least every 2 weeks at Cologne (km 685) over a period of 20 months. In addition, Lagrangian sampling was carried out 160 km downstream of Cologne (Kalkar–Grieth, km 845) over a period of 12 months. Potential HNF growth rates and loss rates caused by planktonic predators were estimated in the laboratory (size fractionation experiments) and compared with the changes in HNF‐density in a water parcel flowing downstream. 2. Mean abundance (±SD) ranged from 7 ± 6 to 4890 ± 560 individuals mL^–1 and was positively correlated with discharge. Heterotrophic nanoflagellates abundance increased up to 30‐fold during flood events, whereas there were only minor changes in the taxonomic composition. 3. HNF growth rate ranged from 0.16 ± 0.12 to 1.98 ± 0.10 day^–1. Between 0 and 95% (mean: 32%) of the gross production was lost to planktonic predators; a larger portion between 0 and 195% (mean: 64%) of the HNF gross production was lost by other means. 4. There may be an important role for benthic predators in the control of pelagic HNF. First, production of HNF was high and grazing losses to planktonic predators low at times when HNF abundance was low. Secondly, high in situ loss rates (not explicable by planktonic predators), which were positively related to temperature, indicate the importance of biotic interactions. Thirdly, the dependence of HNF abundance on discharge indicated a decrease grazing intensity with rising water levels (increase in water volume/colonised river bed ratio). 5. The impact of discharge on planktonic HNF mediated by the grazing impact of benthic predators was modelled, showing a good fit with the field data.     

Dynamics of microbial planktonic food web components during a river flash flood in a Mediterranean coastal lagoon

Episodic river flash floods, characteristic of Mediterranean climates, are suspected to greatly affect the functioning of microbial food webs. For the first time, the abundance, biomass and diversities of microbial food web components were studied before and during 4 consecutive days after a flash flood that occurred in November 2008, in the surface waters of five stations along a salinity gradient from 20 to 36 in the Thau lagoon. Eukaryotic pico- and nanophytoplankton were discharged from the river into the lagoon and increased by 30- and 70-fold, respectively. Bacteria increased by only 2-fold in the lagoon, from around 4–8 × 10⁶ cells ml⁻¹, probably benefiting from river nutrient input. Chlorophyll a increased 4-fold, and pigment biomarkers showed that the dinophyceae, prasinophyceae and prymnesiophyceae were sensitive to the flood perturbation, whereas the bacillariophyceae, cryptophyceae and chlorophyceae were resistant and/or transported to the lagoon from the river. Predator responses were more complex as total heterotrophic flagellate abundance decreased slightly, whereas those of specific naked ciliates increased, particularly for Uronema sp. The flood also induced a specific change in diversity, from a community dominated by Strobilidium spiralis to a community dominated by Uronema sp. The tintinnid community was particularly sensitive to the flood event as the abundance of all species decreased greatly. The high increases in biomass, mainly brought by the river during the flood, could have eventually sedimented to the benthic layer and/or been transported further into the lagoon, supporting the pelagic food web, or have even been exported to the Mediterranean Sea.     

Effects of a natural flood disturbance on species richness and beta diversity of stream benthic diatom communities

Natural hydrological disturbances in streams may reduce biomass and species richness and change community composition within streams. Disturbances can also affect beta diversity among streams if their effects are species specific or vary across sites. We investigated the effect of a natural flood on species richness, community composition and among-streams beta diversity of benthic diatoms (total community and three functional groups: low profile, high profile and motile) of seven streams in New Zealand. Sampling occurred shortly before, 10 days after and 40 days after the flood. Species richness of the total diatom community did not change after the flood. The high-profile group was the only one whose species richness declined after the flood, whereas species richness of the low-profile group increased. Community composition changed after the flood, mostly as a result of species replacement rather than richness differences over time. Finally, among-streams beta diversity did not change after the flood, suggesting that variation in species composition of benthic diatoms among streams may be maintained in the face of flood disturbances.     

Seasonal succession of ciliates in lake constance

We found a recurrent seasonal pattern in abundance and composition of planktonic ciliates in Lake Constance, FRG, over a three-year period. Abundance peaks occurred in early spring and summer/autumn, while ciliate numbers were low in late spring (clear-water phase) and winter. Prostomatida and Oligotrichida dominated in early spring. They responded immediately to the phytoplankton spring bloom, while Haptorida, Peritrichida, and large Scuticociliatida (Histiobalantium) were delayed by 1 to 2 weeks. The spring community broke down at the onset of the clear-water phase.Pelagohalteria viridis containing symbiontic algae appeared shortly after this event. A highly diverse community was recorded in summer/autumn. Peritrichida, small Oligotrichida, and large Scuticociliatida reached their maxima during this season. Small Scuticociliatida were rare throughout the year and contributed moderately to total ciliate numbers only during the cold season. The observed seasonal sequence of pelagic ciliates in Lake Constance is discussed in relation to simultaneously collected data on potential food organisms and grazers.     

Aquatic metabolism short-term response to the flood pulse in a Mediterranean floodplain

The pulsing of river discharge affects biodiversity and productivity of whole river–floodplain ecosystems, triggering the transport, storage and processing of carbon. In this study we investigate the short-term changes in water chemistry and net pelagic metabolism (NEP) in two floodplain lakes in response to a flood pulse. The two oxbow lakes investigated in the floodplain of the Mediterranean Ebro River (NE Spain) showed a clear temporal shift in their metabolic balance, controlled by the river discharge and associated changes in water physical and chemical characteristics. Water chemistry (turbidity, water organic matter, chlorophyll a and nutrients concentration) returned to pre-flood values after 4 days, highlighting the resilience of the ecosystem to flood pulses. Lake NEP was depressed before and during floods to a minimum of −34 mg O₂ m⁻³ h⁻¹, and increased after the flood pulse to a maximum of +463 mg O₂ m⁻³ h⁻¹. The phytoplankton assemblage showed before and after floods a replacement of autotrophic species (e.g. Chlorophyceans) by mixotrophic organisms (e.g. Cryptophyceans, Euglenoids). A linear mixed effects model identified abiotic factors, particularly temperature and river discharge, as significant predictors of the net aquatic metabolism and community respiration during flood conditions. Our results suggest that the role of the Ebro floodplain lakes as sources or sinks of C is complex and relative to the time scale investigated, depending strongly on the river discharge dynamics and the transport of limiting nutrients (phosphorus).     

西江下游浮游植物群落周年变化模式

西江肇庆段是珠江干流汇入珠三角河网水域的咽喉通道,对2009年该江段的浮游植物群落组成及变化进行系统阐析.调查期间共发现浮游植物7门,245种(包括变种、变型),其中硅藻和绿藻是浮游植物群落组成的最主要类群,分别占总种数的42.44％和34.69％,其次是裸藻和蓝藻.PCA分析结果显示,浮游植物各类群的种类丰富度和生物量的周年变化趋势主要受水温和径流量的影响.浮游植物种类丰富度的周年变化呈现明显的高温季节高,低温季节低的特征,除因大多数藻类物种直接喜好高温之外,径流量的增大有助于真浮游绿藻种类的外源注入及半浮游和偶然性浮游硅藻种类的增加.浮游植物生物量的周年变化呈现明显的双峰型,峰值分别出现在8月和11月.然而8月份第1个高峰出现之前,生物量波动不大,尽管水温的升高有助于生物量的增加,但是径流量增大所带来的稀释作用掩盖了水温上升对浮游植物生长的促进作用.真浮游植物生物量的变化趋势和数值与总浮游植物极其接近,主要得益于真浮游硅藻物种颗粒直链藻在总种群中的绝对优势地位.综上,水温升高对浮游植物种类丰富度和生物量的增长均有促进,径流量的增大虽然有助于种类丰富度的增加,但不利于生物量的增加.     

Response of phytoplankton and zooplankton communities in six reservoirs of the middle Missouri River (USA) to drought conditions and a major flood event

We assessed if the qualitative and quantitative aspects of plankton composition in reservoirs of the middle Missouri River were influenced by hydrologic variability. Phytoplankton and zooplankton communities in six reservoirs of this highly regulated system were sampled between 2004 and 2011 during historic drought, subsequent recovery, and a 100-year flood event. The reservoir system encompasses a broad latitudinal gradient of decreasing depth, decreasing water residence time and increasing trophic state. Phytoplankton communities of the upper three reservoirs were co-dominated by planktonic and meroplanktonic diatoms during the drought, recovery, and flood periods, but the proportion of more silicified meroplanktonic diatoms increased in the lower three reservoirs as water residence time decreased. Peak phytoplankton biovolume usually occurred during spring/early summer and was associated with increased hydrologic inflows and outflows. Zooplankton biomass of the reservoir system was dominated by Daphnia spp., but all zooplankton groups decreased as inflows and outflows accelerated during the recovery and flood periods. Rotifer abundances were higher under turbulent conditions associated with dam operations. Canonical correlation analyses suggested that temperature, water residence time, station depth, and water clarity explained more variance in the structures of phytoplankton and zooplankton communities than bioavailable nutrient parameters.     

Effect of biofilm age on settlement of Mytilus edulis

Biofilm ageing is commonly assumed to improve mussel settlement on artificial substrata, but the structure and taxonomic composition of biofilms remains unclear. In the present study, multi-species biofilms were characterized at different ages (1, 2, and 3 weeks) and their influence on settlement of the blue mussel, Mytilus edulis, was tested in the field. As biofilms can constitute a consistent food resource for larvae, the lipid quality, defined as the proportion of related essential fatty acids, may be a selection criterion for settlement. Overall mussel settlement increased on biofilms older than 1 week, and the enhanced settlement corresponded to the abundance and composition of the biofilm community, rather than to essential fatty acid levels. However, during a pulse of phytoplankton, the positive influence of biofilm was not detected, suggesting that pelagic cues overwhelmed those associated with biofilms. The influence of biofilms on mussel settlement could be more crucial when planktonic resources are limited.     

Spatial and temporal variability of surface water in the Kuroshio source region, Pacific Ocean, over the past 21,000 years: evidence from planktonic foraminifera

The Kuroshio Current is the major western boundary current of the North Pacific Ocean and has had a large impact on surface water character and climate change in the northwestern Pacific region. The Kuroshio Current becomes a distinctive surface flow in the Ryukyu Arc region after diverging from the North Equatorial Current and passing through the Okinawa Trough. Therefore, the Ryukyu Arc area can be called the Kuroshio source region. We reconstructed post-21-ka time–space changes in surface water masses in the Ryukyu Arc region using 15 piston cores which were dated by planktonic δ¹⁸O stratigraphy and AMS ¹⁴C ages. Our analysis utilized spatial and temporal changes in planktonic foraminiferal assemblages which were classified into the Kuroshio, Subtropical, Coastal, and Cold water groups on the basis of modern faunal distributions in the study region. These results indicate that the Kuroshio Current and adjacent surface water masses experienced major changes during: (1) the Last Glacial Maximum (LGM), and (2) the so-called Pulleniatina minimum event (PME) from 4,500 to 3,000 yr BP. The Kuroshio LGM event corresponds to severe global cooling and is marked by decreases in planktonic δ¹⁸O values and estimated sea-surface temperature (SST) with the dominance of the Cold water group of planktonic foraminifera. Cooling within the Kuroshio source region was enhanced during the LGM event because the Kuroshio Current was forced eastward due to the formation of a land bridge between Taiwan and the southern Ryukyu Arc which prohibited its flow into the Okinawa Trough. Except for the severe reduction and disappearance of the Pulleniatina group, no clear cooling signal was identified during the PME based on δ¹⁸O values, estimated SST values and variations in the composition of planktonic foraminiferal faunas. The PME assemblages are marked by high abundances of Neogloboquadrina dutertrei, a distinctive Kuroshio type species, along with other species assigned to the Coastal and Central water groups. Subtle ecological differences exist between Pulleniatina obliquiloculata and N. dutertrei; i.e. P. obliquiloculata exhibits lower rates of reproduction under conditions of lower primary productivity in the central Equatorial Pacific Ocean. El Niño-like conditions in the Equatorial Pacific Ocean result in lower rates of surface transport in the Kuroshio Current. In turn, this response triggers lower rates of primary productivity in central equatorial surface waters as well as in the upstream Kuroshio source region, ultimately resulting in a lower abundance of P. obliquiloculata. Thus, we interpret the PME as a possible proxy signal of El Niño-like conditions and enhancement of the El Niño Southern Oscillation climate system after the PME in the tropical and sub-tropical Pacific Ocean.     

Intensified organic carbon dynamics in the ground water of a restored riparian zone

1. River restoration projects usually aim at improving the physical habitat for aquatic organisms. The extent to which biogeochemical processes and microbial activities are intensified in restored river reaches remains uncertain. 2. Here, we investigated the relationships between the distribution and composition of organic carbon (OC), bacterial secondary carbon production and extracellular enzymatic activity (EEA) in the ground water below a restored riparian section of the River Thur, Switzerland, relative to a channelised section. The spatiotemporal variability in the stable C isotopic ratio, dissolved OC polydispersity (the distribution of molecular mass in a mixture of molecules) as well as bacterial abundance, EEA and secondary production were investigated in different process zones. 3. At high river discharge, humic as well as low molecular weight amphiphilic substances infiltrated into the subsurface in a zone dominated by the pioneer plants Salix viminalis (willow bush). Concurrently, bacterial abundance, EEA and secondary carbon production increased at this location. 4. The willow plants leached bioavailable substrates into the ground water when the water table was high. The flood‐driven soil–groundwater coupling stimulated EEA and bacterial secondary production of the suspended groundwater bacterial community. 5. Establishing riparian habitat diversity adds hot spots of OC inputs during flood events, potentially providing valuable ecosystem services (e.g. degradation of organic pollutants) that accompany.     

The effect of lake fertilisation on the rotifers of Seathwaite Tarn,an acidified lake in the English Lake District

Seathwaite Tarn, in the English Lake District, was used as an experimental site to test the feasibility of increasing the pH of an acidified waterbody by adding a phosphorus-based fertiliser solution. The impact of this experiment on the planktonic rotifer community was assessed by monitoring for two years before treatment (1990–1991), and for a further two years once treatment had begun (1992–1993). The pH of the water rose by 0.5 pH units over the period of fertilisation, and the lake became very eutrophic. This had little effect on the species composition of the rotifer assemblage, which continued to reflect the acid, oligotrophic nature of the untreated lake. However, there were marked changes in the levels of abundance of most planktonic species. Population densities of Keratella serrulata and Trichocerca longiseta increased in early spring, compared to pre-treatment levels, apparently due to the rises in chlorophyll-a concentrations. In contrast, Polyarthra dolichoptera became very scarce after fertilisation began. The treatment seemed to have little effect on the population dynamics of the dominant openwater rotifer, Polyarthra remata. In 1993, very high densities of Bosmina coregoni (up to 250 × 10³ ind M^–3 ) developed in response to the eutrophication programme. This seemed to suppress the rotifer community in the summer months.     

Physical factors and dissolved reactive silica affect phytoplankton community structure and dynamics in a lowland eutrophic river (Po river,Italy)

We tested the hypothesis that species composition and persistence of phytoplankton communities in nutrient rich lowland rivers depends mainly on physical factors. The study aimed to analyse the effects of water discharge, temperature and chemistry on phytoplankton dynamic and species composition in the lowland reach of the eutrophic Po river (Italy). Both taxonomical and morpho-functional methods were used. True planktonic and tychoplanktic (i.e. detached taxa of benthic origin that remain in suspension) species were found, among which only a few taxa and functional groups prevailed. Diatoms were the most abundant, with a clear dominance of species either sensitive to the onset of water stratification or well adapted to turbid waters. Phytoplankton abundance, biomass and chlorophyll-a followed similar trends, attaining the highest values in summer, at low discharge rates. Correlation and multivariate analysis revealed that the development of a stable phytoplankton community was mainly controlled by water discharge rates. Namely, changes in water flow rates induced major variations in the community structure. The seasonal succession of phytoplankton assemblages was also related to water temperature and dissolved reactive silica availability to some extent overlapping flow effects.     

广西北部湾茅尾海夏冬季浮游细菌群落结构及其影响因子

海洋浮游细菌在生物地球化学循环过程中具有不可替代的作用。为解析广西北部湾茅尾海夏冬季浮游细菌群落结构及其关键环境影响因子，于2017年6月(夏季)和2017年12月(冬季)在该海域设置7个站点，采集0.5m处表层海水样品，利用16S核糖体RNA (16S rRNA)基因高通量测序技术，分析茅尾海浮游细菌群落结构与多样性。结果表明：茅尾海海域浮游细菌隶属于11个门、36个纲、86个目、188个科和506个属；主要浮游细菌类群为变形菌门(Proteobacteria, 52.47%)、厚壁菌门(Firmicutes, 17.34%)、放线菌门(Actinobacteria, 16.39%)、拟杆菌门(Bacteroidetes, 7.54%)和蓝细菌门(Cyanobacteria, 5.38%),共占浮游细菌总丰度的99.12%。北部湾茅尾海夏季浮游细菌群落Chao 1指数和Richness指数均显著(P<0.05)高于冬季。主坐标分析(PCoA)和相似性分析(ANOSIM)结果表明，不同季节浮游细菌群落结构存在极显著差异(R=0.586,P<0.001)。在目分类水平上进行组...     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

Impact of a flood event on the zooplankton of an estuarine lake

Shallow coastal lakes are prone to large fluctuations in physico-chemical variables such as salinity and turbidity. This is now escalating in response to global change. A flood event in March 2014 resulted in a silt plume spreading through part of Lake St Lucia (South Africa). To determine the impact of this event on zooplankton, the Narrows region of St Lucia was sampled on a monthly basis from March to September 2014. For comparative purposes, data from samples collected prior to the flood event were included in the analyses. Analysis of similarity (ANOSIM) revealed dissimilarities in zooplankton community structure among the sampling occasions. The March 2014–May 2014 period was characterized by the highest abundance of freshwater species. Conversely, the abundance of the resident St Lucia copepods Acartiella natalensis and Oithona brevicornis was lowest during this time, and highest in September 2014. The other dominant copepod Pseudodiaptomus stuhlmanni prevailed in March 2014, but declined markedly in April. As of September 2014, P. stuhlmanni had yet to regain its pre-flood densities. The BIOENV procedure, which relates biological and environmental data, revealed that turbidity, salinity and dissolved oxygen were responsible for the observed changes in zooplankton community structure during the study period. Careful management of turbidity and salinity is stressed, as both factors are major drivers of the biota of St Lucia and similar systems worldwide.     

Biofilm diatom community structure: Influence of temporal and substratum variability

Abstract

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Distribution and abundance of trees in floodplain forests of the Wisconsin River: Environmental influences at different scales

Questions: 1. How do physiography, flooding regime, landscape pattern, land‐cover history, and local soil conditions influence the presence, community structure and abundance of overstorey trees? 2. Can broad‐scale factors explain variation in the floodplain forest community, or are locally measured soil conditions necessary? Location: Floodplain of the lower 370 km of the Wisconsin River, Wisconsin, USA. Methods: Floodplain forest was sampled in 10 m × 20 m plots [n= 405) during summers of 1999 and 2000 in six 12‐ to 15‐km reaches. Results: Species observed most frequently were Fraxinus pennsylvanica, Acer saccharinum and Ulmus americana. Physiography (e.g. geographic province) and indicators of flooding regime (e.g. relative elevation and distance from main channel) were consistently important in predicting occurrence, community composition, and abundance of trees. Correspondence analysis revealed that flood‐tolerant and intolerant species segregated along the primary axis, and late‐successional species segregated from flood‐tolerant species along the secondary axis. Current landscape configuration only influenced species presence or abundance in forests that developed during recent decades. Land‐cover history was important for tree species presence and for the abundance of late‐successional species. Comparison of statistical models developed with and without soils data suggested that broad‐scale factors such as geographic province generally performed well. Conclusions: Physiography and indicators of flood regime are particularly useful for explaining floodplain forest structure and composition in floodplains with a relatively high proportion of natural cover types.     

Case studies of variations in suspended matter transport in small catchments

Changes of water and suspended sediment composition during three single flood events were investigated in two small catchments in the Mosel region. In addition to suspended sediment characteristics (turbidity, loss on ignition, density, chlorophyll content), several different nutrients and heavy metals were determined in the dissolved and particulate phase. The flood events investigated were characterized by a single peaked hydrograph. In contrast suspended sediment concentrations and the other parameters showed more complex behaviour. The transport of suspended sediment was not only controlled by discharge magnitude. In the course of a flood event different suspended sediment sources are activated. Using the chemograph and the timing of samples collected during the hydrograph these suspended sediment sources can be identified. In addition to the remobilization of sediment and channel erosion, inputs of suspended solids from a sewage plant, road discharge, topsoil and interflow could be identified.