Effects of cell-bound microcystins on survival and feeding of Daphnia spp

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.     

Survival of Campylobacter spp. in bovine faeces on pasture

Aims:  To determine the survival on pasture of Campylobacter spp. naturally present in bovine faeces and compare this with a previously published study using laboratory-cultured Campylobacter spp.
Methods and Results:  Ten freshly collected cow pats were deposited on pasture during summer, and Campylobacter spp. were enumerated by enrichment broth culture. The counts in three pats were below detection limits. Counts of Campylobacter spp. in the other seven pats fell below detection limits within 14 days. The geometric means of the counts up to 7 days produced a T ₉₀ of 2·2 days. Characterization of Campylobacter spp. by PCR and pulsed field gel electrophoresis indicated the presence of at least six genotypes of Campylobacter jejuni , Campylobacter coli and Campylobacter lari .
Conclusions:  Campylobacter spp. naturally present in cow faeces exhibited a similar survival rate to that previously determined using laboratory-cultured strains. The highly variable counts of naturally occurring Campylobacter spp., and the predominance of lower counts, also support the earlier decision to use laboratory-cultured strains in survival experiments.
Significance and Impact of the Study:  This study reaffirms the short survival of Campylobacter spp. in cow faeces deposited on pasture. This information will be incorporated into a 'reservoir model' for Campylobacter spp. in cow pats on New Zealand pastures.     

Survival of Azotobacter spp. in Dry Soils

Dry soils stored in glass containers in the laboratory and protected from contamination for periods of 22 to 24 years yielded numerous colonies of Azotobacter chroococcum and other members of the family Azotobacteraceae. These results were compared with those reported in 1974, and the findings are uniformly consistent in terms of surviving populations. The data prove that these bacteria remain viable after prolonged periods of dormancy in much the same way as do the endospores of gram-positive bacteria.     

Feeding behaviour and morphology of filtering combs of Daphnia galeata

The population of Daphnia galeata Sars from the fish pond Velký Pálenec (Blatná, Czechoslovakia) living in high food conditions (7 mgC l^–1) was characterized by a small size of the filtering comb on the thoracic limb 3, measured as seta length, length of the base of the comb and number of setae (population 1). One month cultivation of this population in low food conditions (1.5 mgC l^–1) in the laboratory resulted in twofold increase in size of the filtering comb (population 2). Filtering and ingestion rates of both populations were measured at eight concentrations of food (approximately 0.025–3.2 mgC l^–1) using ¹⁴C labeled Scenedesmus acutus. The results show that size of the filtering combs influences considerably feeding behavior of Daphnia. The comparison of animals with the same body length suggests that the population with a large comb feeds at concentration of food below 0.4 mgC l^–1 more intensively and reaches the maximum of the filtering rate at a lower concentration than the population with a small comb. The situation is opposite at concentration above 0.4 mgC l^–1. The higher values of theoretical flow in population with a small projection of filtering area suggest that this population has to compensate disadvantage of a small comb with the higher appendages beat frequency.     

The effects of global warming on Daphnia spp. population dynamics: a review

Various species of Daphnia usually play a key role in the food web of temperate freshwater systems. There is much evidence to show that climate change may influence Daphnia population dynamics, consequently altering both predator–prey interactions and the efficiency of algal biomass control in these ecosystems. This review will analyse and discuss the current knowledge on Daphnia responses to climate warming based on an analysis of selected papers. The presented results indicate that warming may have important direct and indirect effects on Daphnia biology and ecology via its influence on their life-history processes (metabolism, growth, reproduction) and the properties of their habitats. The plasticity of daphnids in terms of adaptive responses is generally high and includes phenotypic adaptations and changes in genotypes, although it also depends upon the strength of selection and the available genetic variation. The seasonal timing and magnitude of temperature increases are important for seasonal biomass fluctuations of Daphnia and similarly influence the potential synchrony of daphnids and phytoplankton succession (the timing hypothesis). In light of the most recent studies on this topic, even a minor warming during short but critical seasonal periods can cause factors that disturb Daphnia population dynamics to coincide, which may destabilize lake food webs by decoupling trophic interactions. Both winter and spring are important critical periods for determining future seasonal fluxes of Daphnia spp. and, consequently, the time of the clear-water phase and the occurrence and duration of Daphnia midsummer decline. Winter conditions may also affect the impact of fish predation on daphnids during summer months. However, the effects of global warming on Daphnia population dynamics and on ecosystem functioning are often difficult to predict due to their complexity and the presence of both antagonistic and synergistic drivers. Thus, the diverse responses of daphnids to climate anomalies depend on both biotic (predator abundance and seasonal phytoplankton succession) and abiotic factors (e.g. hydrodynamics, intensity and duration of thermal stratification, trophic state or geomorphology) of lakes, which are directly influenced by weather changes. The analysis and quantification of such complex interactions require the involvement of different kinds of specialists and the development of accurate research approaches, such as molecular genetic methods or mathematical modelling.     

Feeding in Daphnia galeata on Oscillatoria limnetica and on Detritus Derived from It

Oscillatoria spp. are generally very abundant in many shallow,eutrophic lakes in the Netherlands. However, this is less truefor Daphnia galeata. The main aim of this study was to investigatewhether the edibility of live Oscillatoria limnetica by Daphniagaleata, and of the detritus derived from this filamentous cyanobacterium,was, among others, an important limiting factor for the daphnids.We measured the consumption and assimilation rates of Daphniausing dual-labelling radio-tracer techniques (¹⁴C and ³²P) tolabel separately the live Oscillatoria filaments and detritusprepared from these filaments. The two food types were mixedin different proportions, and both the food ingestion and foodincorporation rates by daphnids were measured. The main findingsof this study were that specific clearance rates of Daphniaon shorter Oscillatoria filaments were significantly higherthan on the longer filaments, in other words the weight-specificingestion rates were higher on the shorter Oscillatoria filamentsthan on the longer filaments from the batch cultures. The longerOscillatoria filaments are more likely to clump and, therefore,are more liable to be rejected by Daphnia during the food collectionand ingestion processes. The shorter filaments, in comparison,are apparently less prone to clumping and, therefore, are clearedby the daphnids at higher rates than the longer filaments. Feedingthe daphnids on double-labelled (¹⁴C and ³²P) shorter filamentsrevealed that the assimilationefficiency of shorter Oscillatoriafilaments was generally higher for P than for C, probably becauseof a high C/P ratio of Oscillatoria. Daphnia (0.75–1.85mm in size) fed significantly better on dead (detritus) Oscillatoriafilaments than on live Oscillatoria filaments, even if the relativeproportion of detritus in the food was only about one-quarterthat of the live filaments. This preference for detritus overlive Oscillatoria, as indicated by Chesson's selectivity coefficienta, was apparently a passive process, rather than a case of activefood selection. This ‘selective’ feeding on detrituswas apparently facilitated by the relatively greater rejectionof the live filaments than the detrital filaments. At very highfood concentrations (15–25 mg C l^–1), with the shareof the live Oscillatoria filaments 2 to 2.5 times greater thanthat of the detritus, the ingestion rates increased proportionallyless with increase in daphnid size than was expected on thebasis of the allometric relationship between the length andweight of these animals. This implies that the larger animalshad greater interfering effects of the Oscillatoria filamentson the food collection and ingestion processes.     

Survival adaptations of threeEuphorbia spp. in arid ecosystem

Three species ofEuphorbia (E. granulata, E. prostrata andE. hirta) exhibited both morphological and physiological adaptations in dry environmental conditions. High bound water, thick cuticle, dense hair covering and low stomatal index are some of the survival adaptations they developed under water stress conditions of the Indian arid zone.     

Effects of Daphnia on microzooplankton communities

Intact phytoplankton and microzooplankton communities from eutrophicStar Lake were incubated for 4 days with and without Daphniapulex, Daphnia galeaia mendotae, or a natural assemblage ofDaphnia species. They were sampled at the onset and terminationof the experiment for bacterial, phytoplankton, ciliate, rotifer,copepod and cladoceran densities. The cladocerans had variedeffects on the rotifers, ranging from significant suppressionof most rotifer species (Keratella cochlearis, Polyarthra remata,Keratella crassa) in the D.pulex jars, to the suppression ofone (K.crassa) or no species in the D.galeata mendotae and StarLake Daphnia assemblage jars, respectively. Small ciliates (<30µm, longest dimension), such as Strobilidium sp. and Pseudo-cyclidiumsp., were adversely affected by most of the cladoceran treatments,while several larger ciliates (>81 µm) were unaffectedin all such treatments. Ciliates were not consistently morevulnerable to cladoceran suppression than similarly sized rotifers.The suppression of ciliates and rotifers was attributable toboth direct effects (predation, interference, or both) and indirecteffects (e.g. resource competition) of the cladocerans. ¹Present address: Department of Biology, University of Louisville,Louisville, KY 40292, USA     

Some Effects of Light on the Growth,Molting, Reproduction and Survival of the Cladoceran,Daphnia pulex

Daphnia pulex were raised under nine light intensities, 0, 1.7, 3.5, 7, 14, 28, 55 and 110 ft-c, polarized light, 6.6 ft-c, and four wavelength ranges, violet, blue, green and red.Of the nine light intensities studied, only 28 ft-c significantly depressed growth. Light generally may retard growth between molts. There was no intensity effect on molting rate although there was a significant affect on the number of preadult molts. Light may affect the amount of time prior to the release of the first brood. Light intensity did not affect the average number of broods per adult but significantly affected the number of young per brood and young per adult and the effects varied. High light intensity significantly affected the number of undeveloped young and nonviable eggs and abortion rate. Reproduction curves under darkness, 3.5, 7, 14, 28 and 55 ft-c do not drop rapidly after an initial peak and apparent plateaus exist with a decline in reproduction during later instars. Light did not significantly affect survival.All effects of polarized light (6.6 ft-c) were indistinguishable from the effects of light intensity.Red wavelengths significantly retard total growth and green wavelengths significantly stimulate molting and retard growth between molts. There was no effect of wavelength on the number of preadult molts but red populations took significantly longer to release the first brood. Wavelengths did not affect the number of broods per adult but blue wavelength significantly increased the number of young per brood and per adult. Wavelength did not affect the number of undeveloped young, nonviable eggs or abortion rate. Reproduction curves under vioîlet and green wavelength do not decline after an initial peak. Animals raised under only a portion of the visible spectrum, and specifically green wavelenghts, had an increased mortality. The effects of wavelengths are probably independent of light intensity.This paper is part of a Ph.D. Thesis submitted to the Faculty of the Graduate School of The University of Kansas.Supported in part by a NSF Summer Traineeship and Grant # 1175 from the University of Kansas Computation Center.Supported in part by a NSF Summer Traineeship and Grant # 1175 from the University of Kansas Computation Center.     

Survival of Escherichia coli and Salmonella spp. in estuarine environments

Survival of Escherichia coli and Salmonella spp. in estuarine waters was compared over a variety of seasonal temperatures during in situ exposure in diffusion chambers. Sublethal stress was measured by both selective-versus-resuscitative enumeration procedures and an electrochemical detection method. E. coli and Salmonella spp. test suspensions, prepared to minimize sublethal injury, were exposed in a shallow tidal creek and at a site 7.1 km further downriver. Bacterial die-off and sublethal stress in filtered estuarine water were inversely related to water temperature. Salmonella spp. populations exhibited significantly less die-off and stress than did E. coli at water temperatures of less than 10 degrees C. Although the most pronounced reductions (ca. 3 log units) in test bacteria occurred during seasonally warm temperatures in the presence of the autochthonous microbiota, 10(2) to 10(4) test cells per ml remained after 2 weeks of exposure to temperatures of greater than 15 degrees C. Reductions in test bacteria were associated with increases in the densities of microflagellates and plaque-forming microorganisms. These studies demonstrated the survival potential of enteric bacteria in estuarine waters and showed that survival was a function of interacting biological and physical factors.     

Survival and detection of Bacteroides spp., prospective indicator bacteria.

Preliminary experiments were performed to assess the use of intestinal Bacteroides spp. as indicators of fecal contamination of water. Viable counts of Bacteroides fragilis, an anaerobic bacterium, declined more rapidly than those of Escherichia coli and Streptococcus faecalis. However, a fluorescent antiserum prepared against B. fragilis successfully detected high proportions (18 to greater than 50%) of B. fragilis cells suspended for 8 days in aerobic water in dialysis bags at the ambient temperature. These percentages were higher than the percent viable recoveries of the two indicator bacteria used for comparison. Thus, the fluorescent antiserum test for B. fragilis might serve as a useful indicator of fecal contamination of water. An advantage of this approach over coliform analysis is the rapidity at which the test can be performed.     

Impact of Violacein-Producing Bacteria on Survival and Feeding of Bacterivorous Nanoflagellates

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.     

Effects of Carrier and Temperature on Survival of Rhizobium spp. in Legume Inocula: Development of an Improved Type of Inoculant

The effects of inoculant carrier, temperature, and storage period on the survival of Rhizobium strains were determined by plate count and most-probable-number analyses. Preliminary experiments showed that survival of rhizobia was affected by each of these factors and their interactions. Results of further studies indicated that six strains of rhizobia survived better at high temperatures when lyophilized and suspended in an oil carrier as compared to finely ground peat. The oil base inocula contained ca. 10⁵ viable rhizobia per g after 56 days of incubation at 60°C, whereas peat base inocula contained ≤10 rhizobia per g. These results suggest that an oil carrier will protect rhizobia from rapid death at usually lethal high temperatures.     

Cell-Bound Lipase and Esterase of Brevibacterium linens

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.     

Role of Microcystins in Poisoning and Food Ingestion Inhibition of Daphnia galeata Caused by the Cyanobacterium Microcystis aeruginosa

The effects of microcystins on Daphnia galeata, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain Microcystis aeruginosa PCC7806 was compared with a mcy⁻ PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to D. galeata, whereas the mcy⁻ mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the Daphnia ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.     

Influence of Culture Conditions on Growth and Survival of Conidia of Trichoderma spp. Coated on Seeds

Five Trichoderma strains were grown on rice, on vermiculite plus potato-dextrose broth (PDB), on potato-dextrose agar (PDA) or in liquid cultures supplemented with glycerol, KCl or polyethylene glycol (PEG) at -1 MPa or - 2 MPa. Conidia were coated on seeds through a methyl cellulose coating or through an industrial film-coating process. The conidial yield decreased with glycerol, KCl or PEG compared with PDB alone. The percentage viability was from 23 to 44% after methyl cellulose coating, regardless of the culture conditions for conidial production. In general, the industrial coating resulted in lower numbers of living conidia. The viability during storage was enhanced when vermiculite, rice or PDA were used as substrates for fungal growth. Nevertheless, temperature of storage was found to be more critical to spore survival than the substrate used for spore production; conidial viability on seeds did not exceed 4 months at 15 C. Solid and liquid cultures produced conidia able to control R. solani and P. ultimum when applied to seeds through industrial film coating. The level of disease suppression varied with the number of viable conidia/seed and with the culture medium used for conidial production. The three main conditions for further industrial application-high yields, longevity and biocontrol effectiveness-might be optimized by selecting the appropriate medium (liquid or solid), water potential and solutes used.     

Population dynamics of Daphnia spp. and implications for trophic interactions in small, monomictic lake

In Lake Oglethorpe, Georgia, USA, herbivorous crustacean zooplankton are abundant and dominate zooplankton biomass in winter, but are scarce throughout most of the summer. We used a 3.5 year study of Daphnia population dynamics to infer when food, predators or temperature constrained growth of this population. Transitions between winter and summer consumer assemblages are concurrent with seasonal changes in water temperature, thermal structure (stratification/destratification), resources (autotrophic/heterotrophic-domained production), and predator abundance and activity (e.g. Lepomis macrochirus and Chaoborus punctipennis. We sampled at weekly or less intervals from April 1992 to September 1995, and determined population abundances for all cladoceran species. For the Daphnia population (Daphnia ambigua + Daphnia parvula, we measured clutch size and length for all individuals. We used average water column temperature (where dissolved oxygen is >1 mg l^-1) to estimate egg development time from an empirical model. Estimates of Daphnia population birth and death rate were thus generated from abundance, egg ratio and temperature/dissolved oxygen data. We compared observed birth rate (bobs) with expected birth rate (bexp>95% CI; predicted for food-saturated conditions at ambient temperature). For variable (1-13 week) periods between later November and March, 1992-1995, water temperature was the primary factor constraining Daphnia population growth (bobs = bexp). From about April to early November, bobs < bexp suggested food-limited population growth. In spring, summer and early fall (March-October), population densities were several-fold lower than in late fall and winter (November-February). However, all else being equal, egg ratio and population birth rate data would have predicted that Daphnia abundance fluctuates over equivalent ranges in spring and fall. We interpret this discrepancy as evidence for increased rates of extrinsic mortality during the growing season and a seasonal shift in the relative importance of resource and predator regulation. The duration of predator suppression of crustacean population abundance in Lake Oglethorpe and other warm-latitude lakes (36N-27°S) is longer (3-7 months) than that observed in north temperate lakes (1-2.5 months; 41-52°N).      

Effects of river sediment elutriates on Daphnia magna

Laboratory sediment bioassays, both acute and chronic, were conducted on sediment elutriates from the River Eurajoki, SW Finland. The river is under impacts of industrial discharges, community effluents and agricultural run-offs. The water flea Daphnia magna Straus was used as a test organism. None of the elutriates tested were acutely toxic. In a long-term bioassay sediment elutriates had effects on survival, body growth and reproduction. Elutriates prepared from samples of a downstream station were characterized by low survival, small body size, reduced total offspring production, and delayed start of reproduction.     

Survival of Escherichia coli, Enterococci, and Campylobacter spp. in sheep feces on pastures

The survival of enteric bacteria in 10 freshly collected sheep fecal samples on pastures was measured in each of four seasons. Ten freshly collected feces were placed on pasture, and concentrations of Escherichia coli, enterococci, and Campylobacter spp. were monitored until exhaustion of the fecal samples. In all four seasons, there was an increase in enterococcal concentrations by up to 3 orders of magnitude, with peak concentrations recorded between 11 and 28 days after deposition. E. coli concentrations increased in three out of four seasons by up to 1.5 orders of magnitude, with peak concentrations recorded between 8 and 14 days after deposition. The apparent growth of E. coli and enterococci was strongly influenced by the initial water content of the feces and the moisture gained during periods of rehydration following rainfalls. Conversely, the results suggested that dehydration promoted inactivation. Campylobacter spp. did not grow and were rapidly inactivated at a rate that tended to be faster at higher temperatures. Pulsed-field gel electrophoresis (PFGE) of a selection of Campylobacter spp. suggested that these survival data are applicable to a range of Campylobacter spp., including the most frequently isolated PFGE genotype from sheep in New Zealand, and to genotypes previously observed to cause disease in humans. The results of this study are currently being incorporated into a fecal microbe reservoir model that is designed to assist water managers' abilities to estimate microbial loads on pastures grazed by sheep, including the influence of factors such as rainfall and temperature.