The dynamics of heterotrophic nanoflagellates and bacterioplankton in a large ultra-oligotrophic Antarctic lake

The abundance of both heterotrophic nanoflagellates (HNAN) andbacterioplankton in a large (9km²) ultraoligotrophic Antarcticlake (Crooked Lake) were investigated from December 1992 untilNovember 1993. HNAN abundance peaked in spring, summer and autumn,falling to lowest numbers during the winter. Numbers rangedbetween 0 and 50.9x10⁴ l^–1. Bacterioplankton abundancewas highest during the late summer and then fell progressivelytowards winter and autumn (range 1.19–4.46x10⁶ l^–1)In contrast to numbers, mean cell volumes (MCV) of the bacteriareached their highest in spring, and consequently highest bacterialbiomass occurred at this time. MCV ranged between 0.052 and0.224µm³. Bacterial production measurements followingthe incorporation of [³H] thymidine into DNA and [¹⁴C] leucineinto protein using a doubling-labelling procedure were undertakenin January, June, August, October and November. Rates variedbetween 2.8 and 52 ng C l¹ h¹. On occasions, a significant differencein production rates based on the uptake of leucine and thymidinewas observed, suggesting unbalanced growth. Highest rates ofproduction coincided with times of high dissolved organic carbonlevels in the water column and lowest production with low levelsof DOC. HNAN grazing rates were measured by following the uptakeof fluorescently labelled bacteria and averaged 4.8 bacterialcells individual¹ day¹ at 2 and 4°C. Specific growth rates(h¹) ranged around 0.00070–0.00077 in both the field andlaboratory, giving doubling times of 37.3 and 41.0 days, respectively.These low rates of grazing and growth indicate that there isno adaptation to low temperatures in these freshwater protists.Based on these data, the gross production efficiency is 24%.HNAN removed between 0.1 and 9.7% of bacterial production perday.     

Seasonal dynamics of free and attached heterotrophic nanoflagellates in an oligomesotrophic lake

1. The seasonal development of heterotrophic nanoflagellates (HNF), bacteria, rotiferans and crustacean zooplankton was studied in the epilimnion of Lake Pavin, an oligomesotrophic lake in the Massif Central of France.
2. HNF abundance varied from 0.1 to 2.5 × 10³ mL^–1. Free-living HNF reached their highest density in spring when the copepod Acanthodiaptomus denticornis dominated the metazooplankton. They were present in low numbers when rotifers and cladocerans were numerous.
3. Attached HNF, consisting of bicoecids and choanoflagellates, were fixed to large diatoms and to the colonial cyanobacterium Anabaena flos-aquae . The abundance of attached HNF was significantly correlated to bacterial abundance, which fluctuated between 1.1 and 2.7 × 10⁶ mL^–1. Highest abundance of these epiphytic protists was recorded when free-living heterotrophic nanoflagellates declined.
4. The comparison of the dynamics of heterotrophic nanoflagellates, bacteria, and the impact of zooplankton grazing suggested that prey abundance, the presence of suitable attachment sites and limited competition from the free-living forms were the main factors controlling the development of the epiphytic flagellate protists. In contrast, the low abundance of free-living forms during the period of rotiferan and cladoceran development suggests the prevalence of a top-down control by predation of the metazoopankton.     

Seasonal dynamics of crustacean zooplankton, heterotrophic nanoflagellates and bacteria in a shallow, eutrophic lake

1. The seasonal development of crustacean zooplankton, heterotrophic nanoflagellates (HNF) and bacteria was examined in Grosser Binnensee, a shallow, eutrophic lake in northern Germany. The grazing impact of Daphnia on bacteria and nanoflagellates was estimated from field data on population abundances and from clearance rates obtained in laboratory experiments. 2. The seasonal succession of zooplankton showed distinct peaks of Daphnia magna, cyclopopid copepods, Bosmina longirostris and Daphnia galeata and D. hynlina. The population dynamics of Dapfinia had the strongest impact on all sestonic components. Daphnia maxima coincided with clearwater phases, and were negatively correlated with particulate organic carbon (POC), HNF and phytoplankton. Bacterial abundance was only slightly affected although daphnids were at times more important as bacterial consumers than HNF, as estimated from measured bacterial clearance rates. Other crustaceans (copepods, Bosmina) were probably of minor importance as grazers of bacteria and nanoplankton. 3. HNF abundance varied from 550 ml^?1 to more than 30000 ml^?1. HNF appeared to be suppressed by daphnids and reached highest densities when copepods dominated the metazooplankton. The variation in HNF abundance was not reflected in the concentration of heterotrophic bacteria, which fluctuated rather irregularly between 5 and 20 ± 10⁶ ml^?1. Long filamentous bacteria which were probably resistant to protozoan grazing, however, appeared parallel to the development of HNF. These bacterial cells, although small in number, could comprise more than 30% of the total bacterial biomass.     

Relationships between heterotrophic nanoflagellates and the demographic response of Daphnia longispina in a eutrophic lake with poor food quality conditions

1. Based on both field data and laboratory studies, the summer population of Daphnia longispina living in a stratified eutrophic lake was examined in relation to the abundance of algae, nanoflagellates and picocyanobacteria.
2. In early July, the Daphnia population replaced Bosmina and remained the dominant zooplankter during summer 1994. Its development in July was concomitant with an increase of edible algae but, despite the apparent abundance in available food, the Daphnia population decreased throughout August suggesting poor food conditions.
3. From mid-August to the beginning of September, the biomass of inedible phytoplankton was greater than that of the smaller, edible fraction. Consequently the average rate of increase, birth rates and fecundity of Daphnia remained low. Although the biomass of heterotrophic nanoflagellates was consistently low, the demographic parameters of Daphnia were correlated throughout this period with these protozoans.
4. Life table experiments run in the laboratory showed that epilimnetic food supported both the growth in length of individual Daphnia and an increase in fecundity, but metalimnetic food supported only individual growth. D. longispina probably failed to reproduce because of the abundance of detritus mixed with the heterotrophic nanoflagellates in the metalimnetic water at that period of the year. The vertical migration of Daphnia into these deeper layers could be caused by a predator avoidance mechanism.     

Changes in cell volume of bacteria and heterotrophic nanoflagellates in a hypereutrophic pond

Changes in cell volume of planktonic bacteria and heterotrophic nanoflagellates (HNF) were examined in a hypereutrophic pond from April to October, 1997. There were marked changes in the abundance of bacteria, HNF and ciliates and in protistan bacterivory during this period. The cell volume of free-living bacteria (0.121 ± 0.031 m³, mean ± SD) was large relative to that reported in the literature. The cell volumes of HNF was 71.1 ± 24.8 m³. Both cell volumes did not follow a seasonal trend. The dominant size class of bacteria was seasonally variable, whereas density of filamentous bacteria was relatively high between August and September. Biomass of filamentous bacteria accounted for up to 33.6% of total bacterial biomass. A correlation analysis for cell volume of bacteria and HNF, density of filamentous bacteria and some microbial variates was performed. The positive correlations detected (p<0.05) were between density of bacteria and cell volume of HNF, and between density of filamentous bacteria and cell volume of HNF.     

Grazing of bacteria and phytoplankton by heterotrophic nanoflagellates in a Baltic Sea sample

Grazing by heterotrophic nanoflagellates on bacteria and phytoplankton was studied in a laboratory experiment, using a natural pelagic community originating from the Tvärminne sea area off the southern coast of Finland. Water was prescreened to remove larger grazers. Four experimental treatments were used: light and dark, with and without added nutrients. The growth of the large heterotrophic flagellates was stimulated by increased production of < 3 m phytoplankton. Clearance rates for heterotrophic nanoflagellates were estimated and were found to be within the range of previously reported values.     

Relative strength of resource and predation limitation of heterotrophic nanoflagellates in a low-productive sea area

The magnitude of resource and predation limitation of heterotrophicnanoflagellates (HNF) was studied in two short-term enclosureexperiments performed in a low-productive sea area in the northernBaltic Sea in 2001. A cross-factorial design was used to simultaneouslyassess the relative importance of the two factors. Resourcelimitation was removed by adding bacteria, and predation limitationwas eliminated by selective filtration. The first experimentwas performed in June just after the spring bloom decline andthe second in September at the end of the productive season.Resource limitation prevailed during both experiments, contributingto 60% of the net growth increase in June and 74% in September.Removal of predators had a significant effect only in June.Evidence for simultaneous resource and predation limitationwas thus found only during the post-bloom situation. The resultswere applied to a model on resource and predation control ofHNF abundances. To evaluate seasonal differences, field dataon HNF and bacteria from a whole year study were applied tothe model. Except for a few occasions during spring, the modelindicated prevailing resource control of HNF at two locationswith slightly different productivity.     

Trophic relationships between planktonic bacteria,heterotrophic nanoflagellates and viruses in a mesoeutrophic reservoir

The abundance, biomass, and production (Р _В) of bacrerioplankton; the taxonomic composition, abundance, biomass of heterotrophic nanoflagellates (HNF) and the rate of consumption of bacteria by HNFs; and the abundance of virioplankton, frequency of visibly infected bacterial cells, virus-induced mortality of bacterioplankton, and viral production were estimated in the mesoeutrophic Rybinsk Reservoir. The rate of bacterial mortality due to viral lysis (7.8–34.1%, on average 17.2 ± 2.0% of daily Р _В) was lower than the consumption of bacteria by the HNF community (15.4–61.3%, on average 32.0 ± 4.2% of daily Р _В). While consuming bacteria, HNFs simultaneously absorbed a significant number of viruses residing on the surface and inside the bacterial cells.     

Differential grazing of two heterotrophic nanoflagellates on marine Synechococcus strains

Grazing of heterotrophic nanoflagellates on marine picophytoplankton presents a major mortality factor for this important group of primary producers. However, little is known of the selectivity of the grazing process, often merely being thought of as a general feature of cell size and motility. In this study, we tested grazing of two heterotrophic nanoflagellates, Paraphysomonas imperforata and Pteridomonas danica , on strains of marine Synechococcus . Both nanoflagellates proved to be selective in their grazing, with Paraphysomonas being able to grow on 5, and Pteridomonas on 11, of 37 Synechococcus strains tested. Additionally, a number of strains (11 for Paraphysomonas , 9 for Pteridomonas ) were shown to be ingested, but not digested (and thus did not support growth of the grazer). Both the range of prey strains that supported growth as well as those that were ingested but not digested was very similar for the two grazers, suggesting a common property of these prey strains that lent them susceptible to grazing. Subsequent experiments on selected Synechococcus strains showed a pronounced difference in grazing susceptibility between wild-type Synechococcus sp. WH7803 and a spontaneous phage-resistant mutant derivative, WH7803PHR, suggesting that cell surface properties of the Synechococcus prey are an important attribute influencing grazing vulnerability.     

Flow cytometric detection and quantification of heterotrophic nanoflagellates in enriched seawater and cultures

A flow cytometric protocol to detect and enumerate heterotrophic nanoflagellates (HNF) in enriched waters is reported. At present, the cytometric protocols that allow accurate quantification of bacterioplankton cannot be used to quantify protozoa for the following reasons: i) the background produced by the bacterial acquisitions does not allow the discrimination of protozoa at low abundance, ii) since the final protozoan fluorescence is much higher than the bacterioplankton fluorescence (more than 35 fold) the protozoa acquisitions lie outside the range. With an increase in the fluorescence threshold and a reduction of the fluorescence detector voltage, low fluorescence particles (bacteria) are beneath the detection limits and only higher fluorescence particles (most of them heterotrophic nanoflagellates) are detected. The main limitation for the application of the cytometric protocol developed is that a ratio of bacteria/HNF below 1000 is needed. At higher ratios, the background of larger cells of bacterioplankton makes it difficult to discriminate protozoa. The proposed protocol has been validated by epifluorescence microscopy analyzing both a mixed community and two single species of HFN: Rhynchomonas nasuta and Jakoba libera. Taking into account the required bacteria/HNF ratio cited above, the results provide evidence that the flow cytometric protocol reported here is valid for counting mixed communities of HNF in enriched seawater and in experimental micro or mesocosms. In the case of single species of HNF previous knowledge of the biological characteristics of the protist and how they can affect the effectiveness of the flow cytometric count is necessary.     

Grazing by ciliates and heterotrophic nanoflagellates on picocyanobacteria in Lago Maggiore, Italy

During the oligotrophication of Lago Maggiore, picocyanobacteria(Pcy) increased in abundance and production. In their bimodal,seasonal cycle, the spring peak was due almost exclusively tosingle cells of Pcy, whereas in late-summer/autumn the varietyof morpho-types increased and larger Aphanothece-like rods appeared.Rates of Pcy cell removal by heterotrophic nanoflagellates andciliates were measured by using fluorescently labelled Pcy (FLC)in four experiments performed during the Pcy population shiftfrom small cocci to larger rods. The ciliate community appearedto be composed mainly of oligotrichs in the first two experiments,and subsequently of scuticociliates; heterotrophic nanoflagellatesdecreased in number from May to September, and there was a sizeshift which might reflect species composition change. Peritrichsemerged as the most efficient Pcy grazers. For the other ciliategroups we observed higher individual ingestion rates duringthe spring experiments than during those performed in late summer/autumn.The heterotrophic nanoflagellates ingested from 0.5 to3 Pcyh^-1 while ciliates ingested from 18 to 80 Pcy h^-1. The grazingimpact of the heterotrophic nanoflagellate community rangedfrom 1.9 x 10³ to 8 x 10³ Pcy ml^-1 h^-1, whereas the ciliatecommunity ingestion rate was one order of magnitude lower (0.2x 10³–0.4 x 10³ Pcy ml^-1 h^-1). A significant inverse correlationbetween Pcy size and the clearance rate of heterotrophic nanoflagellatesand ciliates was found. Our results indicate that protozoa areless efficient in cell uptake when the Pcy are composed of largercells. In Lago Maggiore, the carbon flux from Pcy to protozoadecreased from 29.8 to 10.2 µg C l^-1 day^-1 (May and Septemberrespectively). A tentative balance on an annual basis suggeststhat around 80% of the carbon produced by Pcy is taken up byprotozoa and channelled to metazooplankton.     

Relationship between bacteria, phytoplankton and heterotrophic nanoflagellates along the trophic gradient

Bacterial and heterotrophic nanoflagellates (HNF) abundance, as well as bacterial production and chlorophylla levels, were measured at five sites extending from the coastal zone toward the open Adriatic in the period from March to October 1995. The investigated areas were grouped into trophic categories according to concentrations of chlorophylla. All the biotic-para-meters increased along the trophic gradient, leading to eutrophy, but they did not increase at the same rate. The bacterial biomass: phytoplankton biomass (BB: chla) ratio decreased from about 10 in the very oligotrophic area to 0.8 at the eutrophic site. In contrast, the bacterial abundance: HNF abundance ratio (B: HNF) increased from 1000 bacteria per 1 flagellate in the oligotrophic system to 1700 bacteria flagellate⁴ in the eutrophic area. Decreasing BB: chla and increasing B: HNF ratios along the trophic gradient might reflect the different structures of the microbial food web. Relationships between bacterial abundance and production, and chla and HNF showed that bacterial abundance along the trophic gradient was regulated by the interplay between nutrient supply and grazing pressure. But in the oligotrophic system, bacterial abundance was more closely related to bacterial production and chla than in the eutrophic system, suggesting stronger control of bacterial abundance by substrate supply. On the other hand, the coupling between bacteria and HNF, and uncoupling between bacterial abundance and production in the eutrophic system, showed that the importance of bacteriovory increased in richer systems.     

Particle handling during interception feeding by four species of heterotrophic nanoflagellates

High resolution video-microscopy was used to observe grazing patterns of the heterotrophic nanoflagellates Cafeteria roenbergensis, Bodo saltans, Spumella sp., and Ochromonas sp. Spumella and Ochromonas enclose food particles with pseudopodia while Cafeteria and Bodo engulf particles by invagination of the cell surface. The following parameters of the feeding process were quantified: frequency of flagellar beating, speed of particles in different positions of the feeding current, food size selection, feeding rate, and the time budget for the handling of particles. The mean handling times differed between 94 s for Cafeteria and 4 s for Ochromonas for ingested particles. Handling times for ingested particles were significantly longer than for non-captured particles. Long handling times were calculated to be disadvantageous only for flagellates which propel a high water volume per hour (esp. Ochromonas) or live in a bacteria-rich environment. Our model calculations may provide a reasonable theoretical explanation for a concentration-dependent behavioural variability of the feeding strategy of different heterotrophic nanoflagellates (HNF) species.     

Seasonal and vertical distribution of planktonic bacteria and heterotrophic nanoflagellates in the middle Adriatic Sea

Temporal and spatial patterns of bacteria and heterotrophic nanoflagellates (HNF) were studied monthly from January 1997 to December 1998 in the middle Adriatic Sea. Bacterial and HNF relationships with phytoplankton biomass and temperature were analyzed to examine how the relative importance of bottom-up and top-down factors may shift over seasons and locations. For the coastal area, an inconsistent relationship between bacterial abundance and chlorophyll a and a stronger relationship between bacterial abundance and bacterial production suggest that other substrates than those of phytoplankton origin are important for bacteria. The analysis of simultaneous effects of temperature and bacterial production on bacterial abundance showed that the effect of temperature obscured the effects of bacterial production, suggesting that bacterial growth itself is highly temperature-dependent. The relationship between HNF abundance and bacterial abundance was slightly improved by the inclusion of in situ temperature, bacterial production or both parameters, as additional independent variables. About 60% of the variability in HNF abundance can be explained by bacterial abundance, bacterial production and temperature. In the open sea, tight coupling of bacterial abundance with chlorophyll a concentrations implied that phytoplankton-derived substrates have a dominant role in controlling bacterial abundance. During the colder months, bacterial abundance was high enough to support higher HNF abundance than observed, suggesting that predation exerted a minor depressing influence on bacterial abundance during that period. During the spring-summer period, HNF controlled bacterial standing stocks by direct cropping of bacterial production.Communicated by: H.-D. Franke     

Top–down control of benthic heterotrophic nanoflagellates by oligochaetes and microcrustaceans in a littoral freshwater habitat

1. We investigated trophic interactions between benthic heterotrophic nanoflagellates (HNF) and oligochaetes and microcrustaceans (cladocerans and copepods) transferred from a silty, littoral freshwater habitat to laboratory microcosms. With a newly adapted experimental design we were able to compute (i) predation rates on benthic HNF by the tested metazoan organisms and (ii) growth rates of the natural benthic HNF population when losses because of this predation were excluded. 2. The experiments covered a temperature range of 4–27 °C and a fivefold variation of predator densities (September 2000–February 2002). For 60% of these experiments, significant predation of oligochaetes and microcrustaceans on benthic HNF was revealed. Predation rates on HNF ranged from 0 to 0.256 day^?1. Growth rates of the benthic HNF assemblage varied from ?0.098 to 0.353 day^?1; they were used to estimate the significance of the measured losses in comparison with possible other loss factors. 3. The data suggested that during the major part of the year a high percentage of the HNF production was consumed in the surficial sediment of the investigated system, resulting in a relatively constant and low HNF standing stock and an uncoupling of benthic bacteria and their protistan grazers. Top–down control by microcrustaceans and oligochaetes was identified as one significant, frequently prevailing regulatory factor, while other parameters responsible for the control of benthic HNF densities remain to be examined.     

Patterns in the top-down versus bottom-up regulation of heterotrophic nanoflagellates in temperate lakes

The importance of resources versus that of predators in determiningthe abundance of organisms is modulated by several factors whichvary over time and space. Here, we evaluate the abundance ofheterotrophic nanoflagellates (HNF), the main predators of planktonicbacteria, in the plankton of 16 Quebec lakes as a function ofthe abundance of resources and zooplankton (predators). We analyzethe data at two different scales: among lakes varying in resourcesupply and within lakes. Resource factors (total phosphorus,chlorophyll, bacterial concentration) best explained the abundancesof HNF among lakes differing in resource availability. Whenthe effect of resource availability was removed, the biomassof cladocerans became an important determinant of HNF abundance,with abundance declining and the ratio of bacteria per flagellateincreasing, as the cladocerans increased. Within lakes, HNFabundance was not a direct function of resource concentration.Multiple regression models based on data for many lakes showedthat both bottom-up (food) and top-down (predation) variablespredicted HNF abundance quite well. An examination of the seasonalpattern among individual lakes showed each group of variablesdiffering in importance: bottom-up variables were more importantin spring and top-down variables in midsummer. An examinationof the effects of different assemblages of zooplankton on HNFshowed cladocerans to exert the highest negative impact on HNFand to be responsible for keeping low HNF numbers during mostof the summer with that probably affecting the pathways of energyflux in those lakes dominated by cladocerans versus those dominatedby other zooplankton. The absolute, as well as the relative,importance of the different biotic and abiotic factors examinedin predicting the abundance of HNF is a function of the scaleof the investigation. Among lakes varying greatly in resourcesupply, resources are found to better correlate with HNF abundance.When the analysis is performed at another scale, within lakes,the relative importance of resources and predation in determiningHNF abundances changes depending on the variability superimposedby other factors (seasonality and lake food web structure).     

Effect of heterotrophic nanoflagellates on the loss of virus-like particles in pond water

A decrease in the abundance of virus-like particles (VLP) by heterotrophic nanoflagellates (HNF) was examined using size-fractionated water samples taken from a hypereutrophic pond in December 1999, and in March and July 2000. We recorded a considerable decrease in the abundance of VLP in the 5.0µm filtrate relative to the 0.2–0.8µm filtrates. Decrease rates of VLP were reduced in a parallel 5.0µm filtrate treated with cycloheximide. The loss rates of VLP in 5.0µm filtrate varied in each experiment, and a high rate of loss was found when the growth rate of HNF was high. These results suggested that HNF consumed the VLP and that HNF is an important factor for decreasing viral abundance in freshwater environments.