Development and validation of a quantitative PCR assay for the early detection and monitoring of the invasive diatom Didymosphenia geminata

Didymosphenia geminata is a large, invasive, freshwater diatom that can produce distinctive and robust mucilaginous stalks. Over the last two decades, there has been a worldwide increase in the distribution and severity of D. geminata blooms. These dense, persistent blooms can have severe impacts on native species and ecosystem functioning. D. geminata is usually identified by microscopic methods that are time consuming, resource intensive, and dependent upon expert taxonomic identification, so the extent of surveillance programs has been limited. As an alternative, we have developed a TaqMan quantitative polymerase chain reaction (QPCR) assay for sensitive and rapid detection and enumeration of D. geminata in environmental samples. Species-specific QPCR primers and probe were designed by aligning the D. geminata 18S ribosomal DNA (rDNA) sequence with closely related diatoms. The QPCR assay was linear (R² = 1.00) over a detection range of eight orders of magnitude with a lower limit of approximately two D. geminata cells. QPCR analysis of environmental samples employed the comparative cycle threshold (C_T)-method with an exogenous plasmid used as an internal reference standard. The assay was evaluated using samples collected during a survey of D. geminata in three rivers in the South Island, New Zealand, and from 13 international locations where D. geminata is known to be present. Positive QPCR amplifications were confirmed as the correct amplification product through direct DNA sequencing. Phylogenetic analysis of 18S rDNA sequences suggests that D. geminata is more closely related to species in the family Cymbellaceae rather than Gomphonemataceae as currently classified.     

Phosphorus limitation of the freshwater benthic diatom Didymosphenia geminata determined by the frequency of dividing cells

1. Unlike other nuisance algal species, the freshwater benthic diatom Didymosphenia geminata typically forms blooms in low‐nutrient rivers. The negative association between D. geminata blooming behaviour and nutrient levels appears at both catchment and smaller scales. We conducted a series of trials in streamside experimental channels colonised with D. geminata using water from the D. geminata‐affected, oligotrophic Waitaki River, South Island, New Zealand to determine how elevated nitrate and phosphate concentrations affected D. geminata cell division. Because D. geminata blooms are typically most pronounced in unshaded waters, we also investigated the growth response to shading. In all experiments, we used the frequency of dividing cells (FDC) as a metric of cell division. 2. Concentrations of nitrate and dissolved reactive phosphorus (DRP) in the Waitaki River were very low (4 mg m^?3‐N and <1 mg m^?3 DRP). In pilot trials, substrata colonised by D. geminata were subjected to enrichment by either switching the water source toN‐ and P‐rich spring water or by adding a stock solution. Both trials resulted in periods of rapid cell division lasting at least 8 days. 3. Experimental addition of alone triggered an initial cell division which was not sustained. However, addition of alone or together with resulted in prolonged elevation in cell division indicating that the cell division rate was P‐limited. 4. Reduced light levels resulted in decreased FDC in D. geminata in both ambient and N, N + P and P‐enriched river water. 5. Stimulation of D. geminata division rate by addition of above ambient levels confirms that, while blooming behaviour is often associated with oligotrophic rivers, the cells divide faster with greater levels of phosphorus enrichment.     

Didymosphenia geminata growth rates and bloom formation in relation to ambient dissolved phosphorus concentration

1. The bloom‐forming freshwater stalked diatom Didymosphenia geminata is unusual among algae in that nuisance growths occur almost exclusively in oligotrophic waters. Current hypotheses to explain this phenomenon have assumed supplemental acquisition of phosphorus from novel sources within the stalk/mat matrix. 2. We carried out a synoptic survey of river sites in the South Island, New Zealand, to determine whether D. geminata cell division and stalk development (measured as mat coverage or standing crop) were related to ambient phosphorus concentrations in the overlying river water. 3. High coverage (>50%) by D. geminata was largely concentrated at sites with mean dissolved reactive phosphorus (DRP) <2 mg m^?3 in the overlying water. Didymosphenia geminata was present at only one site with DRP >4 mg m^?3, with very low coverage. Cell division rate (measured as the frequency of dividing cells, FDC) was positively correlated with mean DRP suggesting that division rates were controlled by the available phosphorus concentration in ambient river water. At the same time, FDC was negatively correlated with D. geminata standing crop (measured as an index incorporating percentage cover and mat thickness). 4. In a single river reach with a stable cross‐channel gradient of total dissolved phosphorus (TDP) caused by inflows from a high‐nutrient tributary, again we observed a negative correlation between percentage cover by D. geminata and concentrations of TDP. 5. Finally, we made a series of observations on D. geminata‐dominated communities that had been exposed to water enriched with ‐N and ‐P for 4 weeks, followed by exposure to unenriched water. After 2 weeks of nutrient deprivation, D. geminata cell division rates declined by 60%, mean stalk length increased by 250%, and total carbohydrate quadrupled relative to initial values. The appearance of the community changed from a dark brown mat to a thick pale mat typical of D. geminata blooms. 6. All these results indicate that D. geminata cell division rates are actively controlled by concentrations of available phosphorus in the overlying water and that stalk production (represented by mat thickness and extent, stalk length and total carbohydrate) is inversely related to D. geminata cell division rates. They thus support an explanation for D. geminata blooms in oligotrophic rivers tied to enhanced stalk production in nutrient‐poor waters, rather than through acquisition of additional phosphorus through recycling processes within the mat.     

Didymosphenia geminata (Lyngbye) M. Schmidt (Bacillariophyta) in the Danube basin: New data from the Drava river (northern Italy)

Abstract

The presence of Didymosphenia geminata (Lyngbye) M. Schmidt (Bacillariophyta) was revealed in the Drava river and in its affluent Rio Sesto, both flowing in the Alto Adige/Südtirol region of Italy and belonging to the Danube basin. New data are given on the distribution of the species in the Danube basin.     

Manipulating the intensity of near-bed turbulence in rivers: effects on benthic invertebrates

1. Flow conditions were modified over patches of river bed in three rivers in south-western Australia to determine the effects of turbulence on benthic invertebrate communities.
2. Artificial structures to increase downstream turbulence were developed in a laboratory flume. In the field, these increased turbulence intensity by 35% for a 20% reduction in velocity.
3. Patches of gravel were placed in each river and turbulence-generating structures allocated randomly to half of these, creating treatment patches. An acoustic Doppler velocimeter was used to measure flow conditions over both treatment and control patches at several heights above the bed. After 6 weeks, the invertebrate fauna of the gravel patches were sampled to examine the response to modified flow conditions.
4. The treatments increased relative turbulence intensity twofold for a reduction in velocity of between 3 and 5 cm s⁻¹, but turbulence intensity was significantly higher in only one of the three rivers.
5. There were no significant effects of increased relative turbulence intensity on any aspect of the invertebrate assemblage. This may be a result of the fairly small increase in relative turbulence intensity created during the experiment, the spatial scale of the manipulation or the types of stream community studied.     

Didymosphenia geminata invasion in South America: Ecosystem impacts and potential biogeochemical state change in Patagonian rivers

The diatom Didymosphenia geminata has emerged as a major global concern, as both an aggressive invader of rivers and streams in the southern hemisphere, and for its ability to form nuisance blooms in oligotrophic systems in its native range. South American D. geminata blooms were first documented in Chilean Patagonia in May 2010, and have spread to over five regions and three provinces, in Chile and Argentina respectively. The Patagonian invasion represents a distinct challenge compared to other regions; not only are affected systems poorly characterized, but also a general synthesis of the nature and magnitude of ecosystem impacts is still lacking. The latter is essential in evaluating impacts to ecosystem services, forms the basis for a management response that is proportional to the potentially valid threats, or aids in the determination of whether action is warranted or feasible. Based on a revision of the recent literature, some of the most significant impacts may be mediated through physical changes: substantially increased algal biomass, trapping of fine sediment, altered hydrodynamics, and consequent effects on biogeochemical states and processes such as redox condition, pH and nutrient cycling in the benthic zone. Surveys conducted during the early invasion in Chile show a strong correlation between benthic biomass and associated fine sediments, both of which were one–two orders of magnitude higher within D. geminata blooms. Experimental phosphorous amendments showed significant abiotic uptake, while interstitial water in D. geminata mats had nearly 10–20 fold higher soluble reactive phosphorous and a pronounced pH cycle compared to the water column. A dominant and aggressive stalk-forming diatom with this combination of characteristics is in sharp contrast to the colonial cyanobacteria and bare gravel substrate that characterize many Patagonian streams. The potential displacement of native benthic algal communities with contrasting functional groups, increased primary producer biomass, and fine benthic sediment accumulation, all may have a significant effect on stream nutrient cycling and ecosystem function, in Patagonia and elsewhere where nuisance blooms have been reported.     

Contrasting effects of low‐level phosphorus and nitrogen enrichment on growth of the mat‐forming alga Didymosphenia geminata in an oligotrophic river

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环境因子对河流底栖无脊椎动物群落结构的影响

底栖无脊椎动物是河流生态系统的重要组成部分,在物质循环和能量流动中是不可或缺的重要环节。其群落结构特点与河流环境因子密切相关,能较好地反映河流生态系统健康状况。综述了物理因子(底质、温度、水深、水流、洪水干扰等)、化学因子(溶氧量、p H值、磷、氮等)、生物因子(水生植物、竞争和捕食)、人为干扰(电站建设、城镇化等)和综合因子对河流底栖无脊椎动物群落结构的影响,并根据国内外研究现状指出水流、海拔和洪水干扰等环境因子对河流底栖无脊椎动物群落结构影响的研究较少或不足,对这些环境因子的研究应是今后河流生态学领域需要着力推进重要内容。深入研究和完善环境因子与底栖无脊椎动物群落结构的关系可为保护底栖无脊椎动物群落、流域水生态系统管理和受损河流生态系统修复提供更为全面的科学依据。     

Characterization of a new species in the genus Didymosphenia and of Cymbella janischii (Bacillariophyta) from Connecticut,USA

Two non-native stalk-forming diatoms that were recently observed in the West Branch of the Farmington River, a tributary of the Connecticut River in Connecticut (USA), are characterized morphologically and barcode marker sequences were obtained for each of them. Cymbella janischii, the dominant stalk-forming species during the summer of 2012, previously had not been found in the northeastern USA. Samples of C. janischii were examined microscopically and used to obtain four sequences of the barcode marker, the V4 region of the 18S rDNA gene. Phylogenetic analysis indicated that the four independent sequences of C. janischii were distinct from, but most closely related to, published sequences of C. janischii from Idaho and C. mexicana from Texas, USA. A second non-native stalk-producing diatom, resembling Didymosphenia geminata, was found in November 2012 – June 2013 and first reported as Didymosphenia sp. Over this period, the observed cells had a compressed morphology and were consistently small compared with D. geminata. Sequences of the V4 region, obtained from three independent direct polymerase chain reactions (PCR) of single cells isolated from the Connecticut samples, indicated a close relationship to three published sequences of D. geminata from Italy, New Zealand and the USA, and to D. siberica and D. dentata from Russia. Frustules of the cells used in the PCR reactions were recovered and examined using scanning electron microscopy, providing a direct link between the observed morphology and sequence data. The morphology of the novel Connecticut Didymosphenia taxon was compared with that of other Didymosphenia taxa, being most similar to D. pumila, D. laticollis, D. grunowii and smaller cells of D. geminata. Didymosphenia sp. had a triundulate morphology with a consistent length of 40–60 µm. Given the unique morphological features of this diatom, it is proposed as a new species, Didymosphenia hullii Khan-Bureau, sp. nov.     

七项河流附着硅藻指数在东江的适用性评估

综合运用因子分析、聚类分析、箱型图分析等统计方法评估了河流附着硅藻生物指数(Biological Diatom Index,IBD)、硅藻营养化指数(Trophic Diatom Index,TDI)、斯雷德切克指数(Sládecˇek’s Index,SLA)、特定污染敏感指数(Specific Polluosensitivity Index,IPS)、硅藻属指数(Generic Diatom Index,IDG)、戴斯指数(Descy Index,DESCY)和欧盟硅藻指数(European Economic Community Index,CEE)在东江流域河流水质评价中的适用性。结果显示:SLA与IPS,CEE显著相关(P<0.05),CEE与TDI不相关,其余指标间均极显著相关(P<0.01)。SLA与13项水质理化指标均无相关性,TDI与含氯度(Cl)显著负相关,CEE与溶解氧(DO)、电导率(Cond.)、总氮(TN)显著正相关(P<0.05),其余指标与氨氮(NH4-N)、pH不存在线性相关,与五日生化需氧量(BOD5)、高锰酸盐指数(CODKMnO4)、亚硝氮(NO2-N)、硝氮(NO3-N),总磷(TP)具极显著相关性(P<0.01)。13项水质理化指标中主成分负荷贡献大于50%的八项理化参数DO、BOD5、CODKMnO4、TN、NO3-N、NO2-N、TP和SiO2将试验区水质分为四组。IPS、IBD、IDG和CEE与水质物化分类一致性较好。IPS、IBD、IDG和CEE的逐步判别分析(引入P=0.20,剔除P=0.25)显示IPS和CEE都只选出了NO2-N,分类判别的正确率分别为55.6%和48.1%;IBD引入了NO2-N,CODKMnO4和DO 3个解释变量,判别正确率74.1%;IDG引入BOD5和NO3-N两个变量,分组正确率63.0%。硅藻群落聚类显示,IBD和IDG在水质物化分类的箱型图中呈现出明显合理的趋势。以上研究表明IBD和IDG硅藻指数最适合用于东江河流水质生物监测与评价。     

Distribution of benthic invertebrates in acid,brown water streams in the South Island of New Zealand

Acid, brown water streams are common on the west coast of the South Island, New Zealand. Acid precipitation is not a significant problem in this region where stream water acidity is brought about by high concentrations of humic substances. The interrelationships between pH, alkalinity, conductivity, DOC and total reactive aluminium were investigated at 45 running water sites. pH (range 3.5–8.1) was strongly correlated with alkalinity (range 0–49 g·m⁻³ CaCO₃) and less strongly with conductivity (range 2.0–9.7 mS·m⁻¹). A strong positive correlation was found between DOC and total reactive aluminium concentration both of which were correlated negatively with pH. In all brown water streams, most aluminium was probably in the non-toxic, organically complexed form. Benthic invertebrate assemblages were examined at 34 sites. Taxonomic richness was not correlated with pH and similar numbers of ephemeropteran, plecopteran and trichopteran taxa were taken from acidobiontic (pH ⩽ 5.5), acidophilic (pH 5.6–6.9) and moderately alkaline (pH ⩾ 7.0) groups of streams. Many species occurred over a wide pH range and had a lower limit of about pH 4.5. The mayfly, Deleatidium occurred at 33 sites and was amongst the five most abundant taxa at 32 of them. The stoneflies, Zelandobius confusus, Austroperla cyrene and Stenoperla maclellani, an elmid, Hydora sp. and a caddisfly, Psilochorema sp. also occurred in over half the streams and frequently were abundant. Few habitat specialists were found. Benthic assemblages were not associated strongly with measured physicochemical factors but streams in close proximity tended to have similar faunas. This suggests that the availability of suitable colonizers sets the limits to species richness and is important in determining the composition of benthic assemblages at a particular locality.     

Seasonal variation in benthic invertebrate recolonization of small-scale disturbances in a subtropical Florida lake

Approximately 11.5% of the littoral zone of a hypereutrophic Florida lake is disturbed by the construction of spawning beds by the cichlid fish, Sarotherodon aurea, during a single spawning season. Simulations of these beds were created during three seasons of the year to test for variation in recolonization rates and patterns in the benthic macroinvertebrate community.The seasonal variation in densities of benthic invertebrate populations suggests a direct relationship between reproductive activity and recolonization rate. Recolonization rates of the numerically dominant species, Polypedilum halterale (Diptera: Chironomidae), were much more rapid in the spring and summer than in the winter. In contrast, Hyalella azteca and Glyptotendipes paripes attained peak population densities during the winter season when Polypedilum was present in low densities. These organisms invaded the disturbed area in unusually high abundances and later declined to background levels. The final result was a winter population with densities comparable to the control (undisturbed) area, but the species composition was different.Similarity between disturbed and control communities during the winter season only reached 67% a month after disturbance, while communities during the warmer months attained nearly 90% similarity in less than 15 days. This lack of similarity during the winter indicates that disturbance, at the appropriate time, may play a role in community organization.     

Inter-annual changes in the benthic community structure of riffles and pools in reaches of contrasting gradient

The inter-annual variation in the structure of the benthic community of riffles and pools was evaluated in contrasting geomorphic settings. The community structure of riffles and pools was a function of habitat, reach gradient, and discharge and was taxon specific. In years of below average peak discharge, riffles had higher taxon richness than pools (66 versus47) but richness was similar between habitats during a year of average discharge (56 versus 54). The percentage composition of oligochaetes and elmid beetles was more variable inter-annually in pools and low gradient reaches than in high gradient reaches. Differences in the percentage of collector-gatherers and scrapers in riffles and pools appeared related to inter-annual differences in discharge regimes. Two components of the annual discharge regime appear to differentially affect the composition of the benthic community in the snowmelt dominated stream studied: the magnitude of the annual peak discharge and the duration and timing of the period of extended high flow.     

Impacts of introduced brown and rainbow trout on benthic invertebrate communities in shallow New Zealand lakes

1. Brown and rainbow trout have been introduced to many inland waters in New Zealand, but research on the impacts on native communities has focused mainly on streams. The purpose of this study was to compare the benthic communities of trout and troutless lakes. Based on previous studies in North America and Europe, we predicted that the benthic biomass, and especially the abundance of large invertebrates, would be lower in lakes with trout as compared to those without. We surveyed the invertebrate fauna of 43 shallow, high‐elevation lakes (26 with and 17 without trout) in four geographic clusters on the central South Island and then conducted a detailed quantitative study of invertebrate biomass and community structure in 12 of these lakes. 2. Benthic community composition and diversity of lakes with and without trout were nearly identical and biomass was as high or higher in the lakes with as without trout. There was no evidence that trout have caused local extinctions of benthic invertebrates. Although the proportional abundance of large‐bodied aquatic was slightly lower in lakes with than without trout, the abundance of several groups of large‐bodied benthic taxa (dragonflies, caddisflies and water bugs) did not differ. 3. Our findings are in contrast to those in North American and Europe where trout introductions into previously troutless lakes have led to declines in the abundance of benthic invertebrates, especially large‐bodied taxa. We propose that the modest effects of trout in New Zealand could be explained by (i) the high areal extent of submergent vegetation that acts as a benthic refuge, (ii) low intensity of trout predation on benthic communities and/or (iii) characteristics of the benthic invertebrates that make them relatively invulnerable to fish predation. 4. Regardless of the relative importance of these hypotheses, our results emphasise that the same invertebrates occurred in all of the lakes, regardless of size, elevation and presence of trout, suggesting habitat generalists dominate the benthic fauna in shallow New Zealand lakes.     

Effects of salinity gradients on benthic invertebrate and diatom communities in a German lowland river

The Lippe is a lowland river located in the Western part of Germany and has been heavily impacted by coal mining activities ever since. Although mining activities significantly decreased during the last decades, the associated discharge of salt-enriched mine water into the river still poses a persistent threat to the local benthic invertebrate and diatom communities. To analyze the effect of salt pollution on invertebrate and diatom species, biological and chemical data were compiled for this study from a publicly available database. Changes in the community composition due to increased salt concentrations were explored by Non-Metrical-Multidimensional Scaling. Indicator species and salinity thresholds for single species and communities were identified using the method TITAN (Threshold Indicator Taxa Analysis). The method is an analytical approach to detect changes in frequency and abundance of species along an environmental gradient by combining the methods of change point analysis (nCPA) and indicator species analysis. The obtained salinity preferences and individual and community thresholds were compared to the literature and existing salinity classifications. For both diatoms and benthic invertebrates, Non-Metrical-Multidimensional Scaling showed a clear split between samples of high and low salinities. Significant salinity thresholds were determined for 50 invertebrate and 58 diatom species of which 23 respectively 18 species were described as ‘reliable’ indicators according to the specifications given by Baker and King (2010). A majority of salt-tolerant indicator organisms were invasive species. For both organism groups, major changes in community composition were detected at a conductivity value exceeding 900 μS/cm. A reduction of the average salinity to below this threshold may have positive effects on the overall species richness and the persistence of sensitive taxa in the river Lippe. Individual and community thresholds may however be data-dependent to a certain degree and subjected to fluctuations considering the potential interdependencies between salinity and additional physico-chemical and environmental parameters (e.g. water temperature, lime content).     

Structural and functional responses of benthic macroinvertebrates to acid precipitation in two forested headwater streams (Vosges Mountains, northeastern France)

From December 1996 to August 1997, beech litter breakdown and stream benthic macroinvertebrate communities were investigated to assess the effects of acidic precipitation on community structure and function in two second-order headwater streams of the Vosges Mountains (NE France). Because of microscale changes in bedrock mineral composition, one of the streams was acidified (mean pH=4.53, mean total Al=421 g.l^-1) and the other circum-neutral (mean pH=7.23, mean total Al=36 g.l^-1). Results showed that both litter breakdown rate and macroinvertebrate community structure were drastically affected under acidic conditions. The rate of leaf litter breakdown decreased by nine times in the acidic stream. Benthic sampling showed that scrapers were totally eradicated and both gathering and filtering collectors were drastically reduced. Such drastic effects appear to be the consequences of the toxicity of acid water including both proton and aluminum toxicity. A decrease in shredder abundance and a shift from the efficient acid-sensitive Amphipoda Gammarus fossarum to acid-tolerant Nemouroidea (mainly Leuctra sp.) was observed in the acidic stream. Our results indicate that freshwater acidification significantly alters the action of shredders processing leaf litter in the acidic stream. Consequently, interactions between structural and functional responses to acidification probably have profound consequences on the efficiency of acidified stream ecosystems, which in return may alter downstream functioning.     

Use of benthic diatom communities to evaluate water quality in rivers of southern Poland

Biological and chemical data were processed to estimate trophic stage and degree of pollution in several streams and rivers in southern Poland. The majority were eutrophic and some of them heavily polluted; only a few were oligo-mesotrophic. The differences in the water quality of the rivers were reflected by different types of diatom community and also by the values for some diatom indices, which were calculated using the latest version of the 'Omnidia' database software. Except for the Sládeček's index, all diatom indices correlated significantly with organic load (COD), oxygen concentration, conductivity and most of the measured ions. Some indices showed a significant negative correlation with trophic level (expressed by NH₄-N and PO₄-P). In general, IPS (Specific Pollution Sensitivity Index) and GDI (Generic Diatom Index) indices gave the best results. Among the investigated diatom communities, only a few taxa indicated oligo-mesotrophy and oligo-β-mesosaprobity. Most of the sites were characterised by a greater relative contribution of eutraphent and tolerant ones as well as α-mesosaprobic and polysaprobic diatoms. This study suggests that the structure of benthic diatom communities and diatom indices, especially GDI, can be applied for monitoring rivers in Poland. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biology,distribution and ecology of <Emphasis Type="Italic">Didymosphenia geminata</Emphasis> (Lyngbye) Schmidt an abundant diatom from the Indian Himalayan rivers

Three years of studies on some Indian Himalayan rivers in Jammu & Kashmir, Himachal Pradesh, and Sikkim revealed the presence of Didymosphenia geminata (Lyngbye) M. Schmidt, a large diatom, hitherto unreported from these areas. This is first report on the occurrence of this diatom in some Indian Himalayan rivers: Kishanganga in Jammu & Kashmir, Ravi in Himachal Pradesh in Western Himalaya and in Teesta river and its two tributaries, Lachen Chhu and Lachung Chhu from Sikkim in the Eastern Himalaya. D. geminata is the most abundant taxon in both suspended and benthic algal communities in these rivers. The species exhibits wide ecological amplitude, allowing it to inhabit both cold and warm waters. We also recorded the exotic brown trout, Salmo trutta fario in these Himalayan rivers. However, in Central Himalayan rivers, Alaknanda, Bhilangana, Mandakini and Dhauliganga in Uttarakhand, where this diatom was absent the trout were absent too. Our studies indicate that D. geminata may extend its distribution range to other Himalayan rivers, hitherto uninhabited by the diatom, depending on the mode of introduction of exotic trout in these rivers.