Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.     

Intraspecific variability in the response of bloom-forming marine microalgae to changed climate conditions

Phytoplankton populations can display high levels of genetic diversity that, when reflected by phenotypic variability, may stabilize a species response to environmental changes. We studied the effects of increased temperature and CO(2) availability as predicted consequences of global change, on 16 genetically different isolates of the diatom Skeletonema marinoi from the Adriatic Sea and the Skagerrak (North Sea), and on eight strains of the PST (paralytic shellfish toxin)-producing dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Maximum growth rates were estimated in batch cultures of acclimated isolates grown for five to 10 generations in a factorial design at 20 and 24°C, and present day and next century applied atmospheric pCO(2), respectively. In both species, individual strains were affected in different ways by increased temperature and pCO(2). The strongest response variability, buffering overall effects, was detected among Adriatic S. marinoi strains. Skagerrak strains showed a more uniform response, particularly to increased temperature, with an overall positive effect on growth. Increased temperature also caused a general growth stimulation in A. ostenfeldii, despite notable variability in strain-specific response patterns. Our data revealed a significant relationship between strain-specific growth rates and the impact of pCO(2) on growth-slow growing cultures were generally positively affected, while fast growing cultures showed no or negative responses to increased pCO(2). Toxin composition of A. ostenfeldii was consistently altered by elevated temperature and increased CO(2) supply in the tested strains, resulting in overall promotion of saxitoxin production by both treatments. Our findings suggest that phenotypic variability within populations plays an important role in the adaptation of phytoplankton to changing environments, potentially attenuating short-term effects and forming the basis for selection. In particular, A. ostenfeldii blooms may expand and increase in toxicity under increased water temperature and atmospheric pCO(2) conditions, with potentially severe consequences for the coastal ecosystem.     

Allelopathic effects of the Baltic cyanobacteria Nodularia spumdigena, Aphanizomenon flos-aquae and Anabaena lemmermannii on algal monocultures

Allelopathy, the release of extracellular compounds that inhibit the growth of other microorganisms, may be one factor contributing to the formation and/or maintenance of cyanobacterial blooms. We investigated the allelopathic effects of three cyanobacterial species (Nodularia spumigena, Aphanizomenon flos-aquae and Anabaena lemmermannii) that frequently form mass-occurrences in the Baltic Sea. We exposed monocultures of three phytoplankton species (Thalassiosira weissflogii, Rhodomonas sp. and Prymnesium parvum) to cell-free filtrates of the three cyanobacteria, and quantified allelopathic effects with cell counts. We also investigated the role of the growth phase of cyanobacteria in their allelopathy, by comparing the effects of an exponential and a stationary phase culture of N. spumigena. All tested cyanobacteria inhibited the growth of Rhodomonas sp., but none of them affected P. parvum. The effects on T. weissflogii were more variable, and they were amplified by repeated filtrate additions compared to a single filtrate addition. N. spumigena was more allelopathic in exponential than in stationary growth phase, whereas the culture filtrate was more hepatotoxic in stationary phase. Hepatotoxins were thus probably not involved in the allelopathic effects, which is also indicated by the allelopathic properties of the non-toxic A. flos-aquae and A. lemmermannii. The results demonstrate that the common Baltic cyanobacteria affect some coexisting phytoplankton species negatively. Allelopathy may therefore play a role in interspecific competition and contribute to cyanobacterial bloom maintenance.     

Multiannual phytoplankton trends in relation to environmental changes across aquatic domains: A case study from Sardinia (Mediterranean Sea)

We investigated multiannual trends in phytoplankton in relation to several environmental drivers. We analyzed ecological data collected during the past two decades from three aquatic sites: an artificial lake, a coastal lagoon, and a marine coastal area. Hydrographic, nutrient, and phytoplankton data were statistically analyzed to detect trends and interactions. In all ecosystems, the chlorophyll a concentration decreased with increasing abundance of small-sized phytoplankton. Phytoplankton dynamics were related to decreased nutrient concentrations in the lake, to dynamics of phosphorus and decreased salinity in the lagoon, and probably to combined top-down and bottom-up processes in the marine gulf.     

Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), a potent virus for mammalian cell gene delivery, possesses an ability to transduce mammalian cells without viral replication. We examined the role of the cellular cytoskeleton in the cytoplasmic trafficking of viral particles toward the nucleus in human hepatic cells. Microscopic studies showed that capsids were found in the nucleus after either viral inoculation or cytoplasmic microinjection of nucleocapsids. The presence of microtubule (MT) depolymerizing agents caused the amount of nuclear capsids to increase. Overexpression of p50/dynamitin, an inhibitor of dynein-dependent endocytic trafficking from peripheral endosomes along MTs toward late endosomes, did not significantly affect the amount of nuclear accumulation of nucleocapsids in the inoculated cells, suggesting that viral nucleocapsids are released into the cytosol during the early stages of the endocytic pathway. Moreover, studies with recombinant viruses containing the nuclear-targeted expression beta-galactosidase gene (beta-gal) showed a markedly increased level in the cellular expression of beta-galactosidase in the presence of MT-disintegrating drugs. The maximal increase in expression at 10 h postinoculation was observed in the presence of 80 muM nocodazole or 10 muM vinblastine. Together, these data suggest that the intact MTs constitute a barrier to baculovirus transport toward the nucleus.     

Plankton community dynamics during decay of a cyanobacteria bloom: a mesocosm experiment

The aim of the work was to study the effects of a decaying cyanobacteria bloom on nutrient dynamics, plankton community development and production rates of bacteria and primary producers. It was hypothesised that the system would turn more heterotrophic following the decay of the bloom. A 10-day outdoors mesocosm experiment was performed in early June in a brackish-water environment. Non-toxic filamentous cyanobacteria Aphanizomenon flos-aquae were added to the treatment, whereas the control lacked cyanobacteria. A. flos-aquae decayed rapidly, and was absent from the units by day 2. Significantly higher bacteria abundances, lower nanoflagellate densities and higher ciliate abundances were found, suggesting a bottom-up regulated process in the treatments bags. N:P ratios were low (6?C12), suggesting N-limitation. Bacteria correlated negatively with numbers of heterotrophic nanoflagellates (HNF), suggesting grazing on bacteria by HNF. Primary production correlated positively with irradiance, chlorophyll a and inorganic nutrients in all units. The rapidly decaying A. flos-aquae biomass imposed a significant bottom-up regulation in the treatment mesocosms, and the system turned from autotrophic into more heterotrophic with time. The rapid decay also caused some similarities and parallel changes between the treatment and the control.     

Allelopathy of Baltic Sea cyanobacteria: no evidence for the role of nodularin

Extracts of Aphanizomenon flos-aquae and Nodularia spumigena,the two most common cyanobacteria forming recurrent blooms inthe Baltic Sea, decrease the abundance of some phytoplanktonspecies via the release of allelopathic substances. We investigatedhow cell-free filtrates of the two cyanobacteria, as well aspurified hepatotoxin nodularin, produced by N. spumigena affectedcell numbers, chlorophyll a content and ¹⁴CO₂ uptake of thecryptophyte Rhodomonas sp. Both cyanobacterial filtrates significantlyretarded the growth of Rhodomonas sp., A. flos-aquae filtrateup to 46%, whereas purified nodularin showed no significanteffect on any of the growth parameters of the cryptophyte. Theseresults suggest that the allelopathic effect of N. spumigenais most probably due to metabolite(s) other than nodularin,possibly acting via the damage of the target cells.     

A Less Saline Baltic Sea Promotes Cyanobacterial Growth,Hampers Intracellular Microcystin Production,and Leads to Strain-Specific Differences in Allelopathy

Salinity is one of the main factors that explain the distribution of species in the Baltic Sea. Increased precipitation and consequent increase in freshwater inflow is predicted to decrease salinity in some areas of the Baltic Sea. Clearly such changes may have profound effects on the organisms living there. Here we investigate the response of the commonly occurring cyanobacterium Dolichospermum spp. to three salinities, 0, 3 and 6. For the three strains tested we recorded growth, intracellular toxicity (microcystin) and allelopathic properties. We show that Dolichospermum can grow in all the three salinities tested with highest growth rates in the lowest salinity. All strains showed allelopathic potential and it differed significantly between strains and salinities, but was highest in the intermediate salinity and lowest in freshwater. Intracellular toxin concentration was highest in salinity 6. In addition, based on monitoring data from the northern Baltic Proper and the Gulf of Finland, we show that salinity has decreased, while Dolichospermum spp. biomass has increased between 1979 and 2013. Thus, based on our experimental findings it is evident that salinity plays a large role in Dolichospermum growth, allelopathic properties and toxicity. In combination with our long-term data analyses, we conclude that decreasing salinity is likely to result in a more favourable environment for Dolichospermum spp. in some areas of the Baltic Sea.     

Bioaccumulation of PSTs produced by Alexandrium ostenfeldii in the northern Baltic Sea

Bioaccumulation of paralytic shellfish toxins (PSTs) produced by the dinoflagellate Alexandrium ostenfeldii was investigated in the northern Baltic Sea. The study was based on the assumption that the toxins released during high magnitude blooms of A. ostenfeldii will accumulate in the biota at the bloom site, especially in bivalves. To test this, experiments with blue mussels (Mytilus trossulus) exposed to toxic A. ostenfeldii in field conditions were carried out together with a field survey aimed to quantify natural distribution of PSTs in the biota. As hypothesized, PSTs accumulated in the tissues of the blue mussels during the incubations. Toxins were also detected in natural bivalve communities at the bloom site, the highest toxin concentrations found in the small Cerastoderma glaucum individuals, exceeding the EC safety limit for shellfish consumption. Relatively high total toxin concentrations were also detected from fish (Perca fluviatilis). These are the first records of PST transfer in the food web of the northern Baltic Sea.