Invertebrates in small shallow lakes and ponds: a new sampling method to study the influence of environmental factors on their communities

Aquatic Ecology - Small shallow lakes (SSL) are reservoirs of biodiversity and provide numerous ecosystem services. Therefore, tools to easily and rapidly assess the biological integrity and...     

High diversity of <Emphasis Type="Italic">Ruppia</Emphasis> meadows in saline ponds and lakes of the western Mediterranean

Saline inland and coastal waterbodies are valuable habitats that deserve attention for the protection of their unique submerged macrophyte beds that render the water clear, stabilize sediments and provide a habitat for high biomasses of invertebrates as food for waterfowl. The ‘continental seagrass’ Ruppia has the widest salinity tolerance among the submerged macrophytes and occurs in a wide variety of saline saltmarsh pond and lagoon systems. Although two cosmopolitan species Ruppia maritima and Ruppia cirrhosa are recognized in Europe and Ruppia drepanensis in the western Mediterranean, their diversity and distribution are not well known. This previously held traditional idea that there are only two widespread Ruppia species suggests a uniform and very homogenized population structure following the hypothesis of long-distance-dispersal through strong bird-mediated dispersal events. Therefore, the Ruppia chloroplast DNA diversity was investigated along a more than 1,000 km transect of the Iberian Peninsula. We studied 492 individuals from 11 wetland areas (17 ponds) and sequenced a 1,753-bp length of seven chloroplast introns. Eight haplotypes represented at least four distinct groups or taxa which is higher than commonly accepted. Six wetland areas contained more than one haplotype and within-pond diversity occurred within distances as small as 30 m (5 out of 17 cases). This underlines the importance of single waterbodies for harbouring haplotypic diversity in Ruppia. Unique haplotypes were observed in four wetland areas and R. maritima was detected only from a low salinity pond, suggesting the species might be more rare than previously accepted. The present results tend to minimize an overall effect of strong bird-mediated dispersal. This emphasizes the role of regional pond habitat diversity for the preservation of Ruppia taxa and their unique haplotype diversity in extreme saline habitats. Guest editors: B. Oertli, R. Cereghino, A. Hull & R. Miracle Pond Conservation: From Science to Practice. 3rd Conference of the European Pond Conservation Network, Valencia, Spain, 14–16 May 2008.     

Evaluation of a variable angle scanning method to estimate relative abundance and distribution of fish using a single-beam echosounder in shallow lakes

The validity of a hydroacoustic procedure was assessed using a combination of horizontal and vertical scanning to map the distribution of targets and to estimate target density in a shallow lake. Three distribution patterns were created using 37–50 artificial targets (metal hex nuts) anchored at known positions. Real and acoustic maps were qualitatively similar. Aggregation indices estimated by hydroacoustics were within 15% of the real values. Target density ranged from 1 to 8 targets per 100 m⁻³. Estimated target densities were within one target of the real values for 88% of our observations. The variable angle approach was used also to monitor daily and seasonal variations in fish distribution and relative abundance outside the littoral zone. Dace Phoxinus eos × P. neogaeus appeared to use the littoral as a refuge during the day and to migrate to the pelagic zone at dusk. The movements of dace outside the littoral zone were limited to the months of June-August. The variable angle acoustic approach can be useful to estimate fish distribution and relative abundance in shallow lakes.     

A new technique to reduce internal phosphorus loading by in-lake phosphate fixation in shallow lakes

A new, flexible, fast, robust and economic technique was developed to treat sediment in shallow lakes with phosphate binding chemicals. The upper 0.15 m of the sediment is thoroughly mixed with ferric chloride using a water-jet manifold coupled to a dosing pump and a navigation control system. Its logistics were tried out in a small, shallow and hypertrophic peat lake, Lake Groot Vogelenzang.     

Molecular analysis of AMF diversity in aquatic macrophytes: A comparison of oligotrophic and utra-oligotrophic lakes

This study aimed to assess AMF diversity in various plant species in lakes with low and relatively high P concentrations to elucidate possible correlations with environmental factors in order for better understanding the functioning of mycorrhizal fungi in submerged plants. A considerable diversity of AMF communities was observed in the lakes with low dissolved P concentrations, especially in the roots of Littorella uniflora. Glomus group A, Archaeospora and Acaulospora were the most frequent and diverse AMF lineages with eight, seven and four phylotypes at Littorella uniflora in at least six lakes with low dissolved P concentrations. In theses lakes, AMF were for the first time observed in the roots of J. bulbosus, a member of a family previously thought to be non-mycorrhizal. In the lakes with relatively high dissolved P concentrations, the frequency decreased from Acaulospora, found at three locations, to Archaeospora at two locations and Glomus group A and Paraglomus at one location.All chemical parameters of the surface water layer, except pH, revealed significant (p ≤ 0.01) differences between the lakes with low and relatively high dissolved P concentrations. Mean Mg²⁺, Ca²⁺, K⁺, NH₄⁺, CO₂, o-PO₄³⁻ and HCO₃⁻ were 3, 13.5, 15.7, 19.5, 31 and 42.6 times higher, respectively, in the lakes with relatively high dissolved P concentrations compared to the lakes with low dissolved P concentrations. AMF occurred more abundantly with low phosphate and high redox values in the lakes than with high phosphate and low redox values. The pH-value, the total-calcium and total-phosphorus concentrations were strongly correlated with the occurrence of Glomus phylotypes 4 and Archaeospora phylotypes 5 and 8, and a bit less with Acaulospora phylotype 4 and Archaeospora phylotype 3. In such lakes the presence of a diverse AMF community still enables the uptake of sufficient P for isoetid plant species despite the prevailing ‘ultra-oligotrophic’ conditions. As a consequence, macrophyte plant communities in lakes with relatively high dissolved P concentrations are less dependent on AMF colonization for their development.     

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.     

Engineering and biological approaches to the restoration from eutrophication of shallow lakes in which aquatic plant communities are important components

Engineering approaches (nutrient removal, sediment pumping, hypolimnion oxygenation, alum treatments) may be most appropriate to deep lakes where the aim of restoration from eutrophication is simply to reduce the production and crop of one component, the phytoplankton. They do not always give the desired results because the nutrient loading may only be reduced to a limited extent. There are additional problems in shallow lakes where change of state between community dominance (aquatic plants versus plankton) is wanted. Each community has powerful buffering mechanisms and biomanipulation may be essential to switch one state to another even with considerable nutrient reduction. For the phytoplankton-dominated community the buffers include the advantages of early growth, lower diffusion pathways for CO₂, overhead shading, and an absence of large cladoceran grazers. This later is because open-water shallow environments provide no refuges against predation for the large Cladocera which are both the most efficient grazers and the most favoured prey for fish. Restoration of aquatic plants may then require provision of refuges for the grazers. Different sorts of refuge are discussed using case studies of Hoveton Great Broad and Cockshoot Broad in the Norfolk Broadland.     

Specific attenuation coefficients of optically active substances and their contribution to the underwater ultraviolet and visible light climate in shallow lakes and ponds

Sunlight penetration through the water column is controlled by the amount and kind of materials dissolved and suspended in the water. Understanding UV penetration in its complexity is essential for the prediction of the impact of UV radiation on aquatic ecosystems. However, only limited data are available on the penetration of UVR into shallow waters rich in inorganic suspended solids and chromophoric dissolved organic matter (CDOM). The same is true for the specific attenuation coefficients of light-absorbing components at the UV waveband. This study analyses the role of CDOM, algal-free suspended solids and algae in the formation of underwater UVR and PAR climate in 30 water bodies from clear gravel pit lakes trough the shallow Lake Balaton to turbid soda pans. Irradiance-depth profiles were obtained at 305, 313, 320 nm (UV-B), 340, 380, 395 nm (UV-A) and 400–700 nm (PAR) with a Biospherical PUV-2500 radiometer. Vertical attenuation coefficients (K _d) were calculated. Water samples were taken for the laboratory measurement of the concentration of light-absorbing components: algae as chlorophyll a (CHL), chromophoric dissolved organic matter as colour (CDOM), and algal-free suspended solids (TSS-Alg) parallel with the in situ light measurements. Specific attenuation coefficient values were calculated by multiple regression analysis (n = 140). The obtained specific UV attenuation coefficient values of CHL, CDOM and TSS-Alg made it possible to establish light attenuation at different wavelengths based on the knowledge of the concentration of these light-absorbing components.     

Sensitive and rapid detection of microcystin synthetase E Gene (mcyE) by loop-mediated isothermal amplification: A new assay for detecting the potential microcystin-producing Microcystis in the aquatic ecosystem

In order to develop a new molecular technique that has the potential to assist with monitoring and management of water bodies for potential microcystin producing cyanobacterial species that occur in mixed populations in many regions of the world, we designed a new loop-mediated isothermal amplification (LAMP) assay based on microcystin biosynthesis genes. Four sets of primers were designed to recognize six distinct sequences on target the mcyE gene that encodes a protein (McyE) being responsible to catalyze the addition of d-glutamate to Adda. One set (MCYE2) was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for mcyE detection were determined. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 40 min at an isothermal temperature of 61 °C. For the sensitivity of LAMP, the detection limit was 8.5 pg/μl (approximately 17 pg) DNA. The eleven microcystin producing and four non-toxic cyanobacterial strains were selected for testing of specificity. The results of the amplification were positive with all microcystin-producing strains tested and not with four non-toxic strains, which showed that the primers had good levels of specificity. For testing the application of LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo City were also analyzed using the LAMP targeting the mcyE gene as well as an ELISA assay. Compared with these results of ELISA assay, LAMP assay is satisfied. All of these validated LAMP method being fast, simple and low in cost is a potentially valuable means for potential toxic of cyanobacterial blooms detection, especially for routine monitoring purposes in future.     

A comparison of the catchment sizes of rivers,streams, ponds,ditches and lakes: implications for protecting aquatic biodiversity in an agricultural landscape

In this study we compared the biodiversity of five waterbody types (ditches, lakes, ponds, rivers and streams) within an agricultural study area in lowland England to assess their relative contribution to the plant and macroinvertebrate species richness and rarity of the region. We used a Geographical Information System (GIS) to compare the catchment areas and landuse composition for each of these waterbody types to assess the feasibility of deintensifying land to levels identified in the literature as acceptable for aquatic biota. Ponds supported the highest number of species and had the highest index of species rarity across the study area. Catchment areas associated with the different waterbody types differed significantly, with rivers having the largest average catchment sizes and ponds the smallest. The important contribution made to regional aquatic biodiversity by small waterbodies and in particular ponds, combined with their characteristically small catchment areas, means that they are amongst the most valuable, and potentially amongst the easiest, of waterbody types to protect. Given the limited area of land that may be available for the protection of aquatic biodiversity in agricultural landscapes, the deintensification of such small catchments (which can be termed microcatchments) could be an important addition to the measures used to protect aquatic biodiversity, enabling ‘pockets’ of high aquatic biodiversity to occur within working agricultural landscapes. Guest editors: R. Céréghino, J. Biggs, B. Oertli & S. Declerck The ecology of European ponds: defining the characteristics of a neglected freshwater habitat     

A new approach to an old conundrum--DNA barcoding sheds new light on phenotypic plasticity and morphological stasis in microsnails (Gastropoda, Pulmonata, Carychiidae)

The identification of microsnail taxa based on morphological characters is often a time-consuming and inconclusive process. Aspects such as morphological stasis and phenotypic plasticity further complicate their taxonomic designation. In this study, we demonstrate that the application of DNA barcoding can alleviate these problems within the Carychiidae (Gastropoda, Pulmonata). These microsnails are a taxon of the pulmonate lineage and most likely migrated onto land independently of the Stylommatophora clade. Their taxonomical classification is currently based on conchological and anatomical characters only. Despite much confusion about historic species assignments, the Carychiidae can be unambiguously subdivided into two taxa: (i) Zospeum species, which are restricted to karst caves, and (ii) Carychium species, which occur in a broad range of environmental conditions. The implementation of discrete molecular data (COI marker) enabled us to correctly designate 90% of the carychiid microsnails. The remaining cases were probably cryptic Zospeum and Carychium taxa and incipient species, which require further investigation into their species status. Because conventional reliance upon mostly continuous (i.e. nondiscrete) conchological characters is subject to fallibility for many gastropod species assignments, we highly recommend the use of DNA barcoding as a taxonomic, cutting-edge method for delimiting microsnail taxa.     

A new method for immunologically marking prey and its use in predation studies

We introduce a new method for immunologically examining predator gut contents. It differs from previously described gut content analyses because it does not require the development of prey-specific antibody probes. Instead, insect prey were marked with a readily available antigen, rabbit immunoglobulin G (IgG). We then assayed predators that had fed on IgG labeled prey with an enzyme-linked immunosorbent assay (ELISA) using goat anti-rabbit IgG. Of the predator species that fed on the IgG labeled prey, 98.8% of those with chewing mouthparts scored positive for IgG 1 h after feeding. Our prey-labeling ELISA was less efficient for detecting IgG prey remains in predators with piercing/sucking mouthparts. Only 29.5% of these individuals scored positive for rabbit IgG in their guts 1 h after feeding. An additional study was conducted to measure the retention time of IgG-labeled prey in the guts of two species of predators with chewing mouthparts. Results from this experiment showed that the retention time varied depending on the predator and prey species examined. Results from these studies indicate that this marking technique could have widespread use for analyzing the gut contents of predators with chewing mouthparts, but it has limited application for those predators with piercing/sucking mouthparts. This article presents the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA.     

On the reliability of a simple method for scoring phenotypes to estimate heritability: A case study with pupal color in Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae)

In this paper, two methods for assessing the degree of melanization of pupal exuviae from the butterfly Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae, Heliconiini) are compared. In the first method, which was qualitative, the exuviae were classified by scoring the degree of melanization, whereas in the second method, which was quantitative, the exuviae were classified by optical density followed by analysis with appropriate software. The heritability (h(2)) of the degree of melanization was estimated by regression and analysis of variance. The estimates of h (2) were similar with both methods, indicating that the qualitative method could be particularly suitable for field work. The low estimates obtained for heritability may have resulted from the small sample size (n = 7-18 broods, including the parents) or from the allocation-priority hypothesis in which pupal color would be a lower priority trait compared to morphological traits and adequate larval development.     

A simple method to estimate the contribution of biological floc and reactor-solution to mass transfer of oxygen in activated sludge processes

In this study, the mass transfer coefficient of biological floc (K(L)a(bf)) was estimated from the mass transfer coefficient of the mixed-liquor (K(L)a(f)) and the reactor-solution (K(L)a(e)). The biological floc resistance (BFR) and reactor-solution resistance (SR) were defined as the reciprocal of K(L)a(bf) and K(L)a(e), respectively, by applying the concept of serial-resistance originally presented in two-film theory (Lewis and Whitman (1924) Ind Eng Chem 16:1215-1220). The specific biological floc resistance (SBFR) was defined as biological floc resistance per unit biomass concentration. The data indicated that an activated sludge process yielding low BFR/MLR and BFR/SR tended to produce higher oxygen transfer efficiency. Surprisingly, the reactor-solution posed the same level of resistance as clean water in all experiments, except in a 5-day SRT, non-nitrifying, completely mixed activated sludge (CMAS) process run. Furthermore, SBFR successfully represented biological floc and showed a positive correlation to sludge volume index (SVI). In addition, SBFR/SR and oxygen transfer efficiency (OTE(f)) followed an exponential relationship for the complete data set. The method of separating the mixed-liquor into biological floc and reactor-solution improved the understanding of oxygen transfer under process conditions, without resorting to intrusive techniques or direct handling of fragile biological floc.     

A new approach to estimate the in situ fractional degradation rate of organic matter and nitrogen in wheat yeast concentrates

In the classic in situ method, small particles are removed during rinsing and hence their fractional degradation rate cannot be determined. A new approach was developed to estimate the fractional degradation rate of nutrients in small particles. This approach was based on an alternative rinsing method to reduce the particulate matter loss during rinsing and on quantifying the particulate matter loss that occurs during incubation in the rumen itself. To quantify particulate matter loss during incubation, loss of small particles during the in situ incubation was studied using undegradable silica with different particle sizes. Particulate matter loss during incubation was limited to particles smaller than ~40 μm with a mean fractional particulate matter loss rate of 0.035 h⁻¹ (first experiment) and 0.073 h⁻¹ (second experiment) and an undegradable fraction of 0.001 and 0.050, respectively. In the second experiment, the fractional particulate matter loss rate after rinsing in a water bath at 50 strokes per minute (s.p.m.) (0.215 h⁻¹) and the undegradable fraction at 20 s.p.m. (0.461) were significantly larger than that upon incubation in the rumen, whereas the fractional particulate matter loss rate (0.140 and 0.087 h⁻¹, respectively) and the undegradable fraction (0.330 and 0.075, respectively) after rinsing at 30 and 40 s.p.m. did not differ with that upon rumen incubation. This new approach was applied to estimate the in situ fractional degradation rate of insoluble organic matter (OM) and insoluble nitrogen (N) in three different wheat yeast concentrates (WYC). These WYC were characterised by a high fraction of small particles and estimating their fractional degradation rate was not possible using the traditional washing machine rinsing method. The new rinsing method increased the mean non-washout fraction of OM and N in these products from 0.113 and 0.084 (washing machine method) to 0.670 and 0.782, respectively. The mean effective degradation (ED) without correction for particulate matter loss of OM and of N was 0.714 and 0.601, respectively, and significant differences were observed between the WYC products. Applying the correction for particulate matter loss reduced the mean ED of OM to 0.676 (30 s.p.m.) and 0.477 (40 s.p.m.), and reduced the mean ED of N to 0.475 (30 s.p.m.) and 0.328 (40 s.p.m.). These marked reductions in fractional degradation rate upon correction for small particulate matter loss emphasised the pronounced effect of correction for undegraded particulate matter loss on the fractional disappearance rates of OM and N in WYC products.     

A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies

Several studies have shown that the fatty acid composition of circulating lipids reflects dietary fat intake, in turn being related to health status. The fatty acid composition of plasma lipids is therefore an important parameter in studies on dietary interventions. The aim of our study was to develop a rapid and inexpensive method for the analysis of circulating fatty acids applicable to large population groups. Drops of blood collected from fingertips have been directly subjected to transmethylation for gas chromatography analysis. This new method, validated for reproducibility, has been compared with the conventional method, based on withdrawal of blood from the antecubital vein followed by lipid extraction, and identical data have been obtained with the two techniques. Observed and predicted differences between blood and plasma fatty acids are related to the contribution of circulating cell membranes in blood. Finally the application of the methods to samples from 100 healthy subjects and the assessed correlation between dietary habits and blood fatty acid profiles demonstrate the validity of the new method and its applicability to nutritional and epidemiological studies.     

A new method to measure intestinal activity of P-glycoprotein in avian and mammalian species

Permeability-glycoprotein (Pgp) actively exports numerous potentially toxic compounds once they diffuse into the cell membrane of intestinal epithelial cells. We adapted the everted sleeve technique to make the first measures of intestinal Pgp function in an avian species (chicken) and in wild mammalian species, and compared them to laboratory rats. Tissues maintained both structural and functional integrity, and our method offers advantages over other in vitro techniques by using smaller intestinal sections (1 cm), and shorter incubation times (8–12 min). To determine Pgp function, we compared accumulation of [³H]-digoxin in sleeves incubated in Ringer solution with and without a transport-saturating concentration of a competitive inhibitor, cyclosporin A. We demonstrated significant variation in Pgp activity within individuals along the intestine, between populations fed different diets, and between species (laboratory rats had one-third to one-fifth the Pgp activity of wild rodents). In chicken, we also tested the effect of natural metabolites on digoxin accumulation. We found that among flavonoids, genistein (200 M), found in soy and other legumes, but not quercetin (10, 30, 100, 330 M) or the 3--glycoside isoquercetrin (100 M), significantly increased digoxin accumulation. Among fungal metabolites, sterigmatocystin (5 M), but not aflatoxin B1 (5 M), significantly increased digoxin accumulation.     

A new, rapid and reproducible method to obtain high quality endothelium in vitro

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals.