Phytoplankton population dynamics are the result of imbalances between reproduction and losses. Losses include grazing, sinking, and natural mortality. As the importance of microbes in aquatic ecology has been recognized, so has the potential significance of viruses as mortality agents for phytoplankton. The field of algal virus ecology is steadily changing and advancing as new viruses are isolated and new methods are developed for quantifying the impact of viruses on phytoplankton dynamics and diversity. With this development, evidence is accumulating that viruses can control phytoplankton dynamics through reduction of host populations, or by preventing algal host populations from reaching high levels. The identification of highly specific host ranges of viruses is changing our understanding of population dynamics. Viral-mediated mortality may not only affect algal species succession, but may also affect intraspecies succession. Through cellular lysis, viruses indirectly affect the fluxes of energy, nutrients, and organic matter, especially during algal bloom events when biomass is high. Although the importance of viruses is presently recognized, it is apparent that many aspects of viral-mediated mortality of phytoplankton are still poorly understood. It is imperative that future research addresses the mechanisms that regulate virus infectivity, host resistance, genotype richness, abundance, and the fate of viruses over time and space.

Over the past two decades, many reports have revealed the existence of bacteria capable of killing phytoplankton. These algicidal bacteria sometimes increase in abundance concurrently with the decline of algal blooms, suggesting that they may affect algal bloom dynamics. Here, we synthesize the existing knowledge on algicidal bacteria interactions with marine eukaryotic microalgae. We discuss the effectiveness of the current methods to characterize the algicidal phenotype in an ecosystem context. We briefly consider the literature on the phylogenetic identification of algicidal bacteria, their interaction with their algal prey, the characterization of algicidal molecules, and the enumeration of algicidal bacteria during algal blooms. We conclude that, due to limitations of current methods, the evidence for algicidal bacteria causing algal bloom decline is circumstantial. New methods and an ecosystem approach are needed to test hypotheses on the impact of algicidal bacteria in algal bloom dynamics. This will require enlarging the scope of inquiry from its current focus on the potential utility of algicidal bacteria in the control of harmful algal blooms. We suggest conceptualizing bacterial algicidy within the general problem of bacterial regulation of algal community structure in the ocean.

Planktonic members of most algal groups are known to harbor intracellular symbionts, including viruses, bacteria, fungi, and protozoa. Among the dinoflagellates, viral and bacterial associations were recognized a quarter century ago, yet their impact on host populations remains largely unresolved. By contrast, fungal and protozoan infections of dinoflagellates are well documented and generally viewed as playing major roles in host population dynamics. Our understanding of fungal parasites is largely based on studies for freshwater diatoms and dinoflagellates, although fungal infections are known for some marine phytoplankton. In freshwater systems, fungal chytrids have been linked to mass mortalities of host organisms, suppression or retardation of phytoplankton blooms, and selective effects on species composition leading to successional changes in plankton communities. Parasitic dinoflagellates of the genus Amoebophrya and the newly described Perkinsozoa, Parvilucifera infectans, are widely distributed in coastal waters of the world where they commonly infect photosynthetic and heterotrophic dinoflagellates. Recent work indicates that these parasites can have significant impacts on host physiology, behavior, and bloom dynamics. Thus, parasitism needs to be carefully considered in developing concepts about plankton dynamics and the flow of material in marine food webs.

For an algal bloom to develop, the growth rate of the bloom-forming species must exceed the sum of all loss processes. Among these loss processes, grazing is generally believed to be one of the more important factors. Based on numerous field studies, it is now recognized that microzooplankton are dominant consumers of phytoplankton in both open ocean and coastal waters. Heterotrophic protists, a major component of microzooplankton communities, constitute a vast complex of diverse feeding strategies and behavior which allow them access to even the larger phytoplankton species. A number of laboratory studies have shown the capability of different protistan species to feed and grow on bloom-forming algal species. Because of short generation times, their ability for fast reaction to short-term variation in food conditions enables phagotrophic protists to fulfill the function of a heterotrophic buffer, which might balance the flow of matter in case of phytoplankton blooms. The importance of grazing as a control of microalgae becomes most apparent by its failure; if community grazing controls initial stages of bloom development, there simply is no bloom. However, if a certain algal species is difficult to graze, e.g. due to specific defense mechanisms, reduced grazing pressure will certainly favor bloom development. The present contribution will provide a general overview on the interactions between planktonic microalgae and protozoan grazers with special emphasis on species-specific interactions and algal defense strategies against protozoan grazers.

The potential use of clays to control harmful algal blooms (HABs) has been explored in East Asia, Australia, the United States, and Sweden. In Japan and South Korea, minerals such as montmorillonite, kaolinite, and yellow loess, have already been used in the field effectively, to protect fish mariculture from Cochlodinium spp. and other blooms. Cell removal occurs through the flocculation of algal and mineral particles, leading to the formation of larger aggregates (i.e. marine snow), which rapidly settle and further entrain cells during their descent. In the U.S., several clays and clay-rich sediments have shown high removal abilities (e.g. > 80% cell removal efficiency) against Karenia brevis, Heterosigma akashiwo, Pfiesteria piscicida and Aureococcus anophagefferens. In some cases, the removal ability of certain clays was further enhanced with chemical flocculants, such as polyaluminum chloride (PAC), to increase their adhesiveness. However, cell removal was also affected by bloom concentration, salinity, and mixing. Cell mortality was observed after clay addition, and increased with increasing clay concentration, and prolonged exposure to clays in the settled layer. Mesocosm, field enclosure, and flume experiments were also conducted to address cell removal with increasing scale and flow, water-column impacts, and the possible benthic effects from clay addition. Results from these studies will be presented, especially those in regards to water quality, seawater chemistry, bottom erodibility and faunal impacts in the benthos. At this time, clay dispersal continues to be a promising method for controlling HABs and mitigating their impacts based on existing information and experimental data.

Studies of benthic Foraminifera typically rely on the morphological identification of dried specimens. This approach can introduce sampling bias against small, delicate, or morphologically ambiguous forms. To overcome this limitation, we extracted total DNA from sediment followed by PCR using group- and species-specific primers. Phylogenetic analyses revealed that approximately ninety percent of the PCR products represented previously undescribed sequence types that group with undersampled members of the allogromiid Foraminifera. We also used a modification of this technique to track individual species in sediment fractions too fine for normal morphological identification, and to confirm species placement of morphologically ambiguous foraminiferans. We were able to identify the DNA of several large foraminiferal species in fine fractions in a seasonally-dependent manner, indicating that in some seasons the majority of the standing stock of these species exists as gametes/juveniles. The approach outlined here represents a powerful strategy for exploring the total diversity of benthic foraminiferal communities.

The phylogenetic relationships among peritrichs remain unresolved. In this study, the complete small subunit rRNA (SSrRNA) gene sequences of seven species (Epistylis galea, Campanella umbellaria, Carchesium polypinum, Zoothamnium arbuscula, Vaginicola crystallina, Ophrydium versatile, and Opercularia microdiscum) were determined. Trees were constructed using distance-matrix, maximum-likelihood and maximum-parsimony methods, all of which strongly supported the monophyly of the subclass Peritrichia. Within the peritrichs, 1) E. galea grouped with Opercularia microdiscum and Campanella umbellaria but not the other Epistylis species, which indicates that the genus Epistylis might not be monophyletic; 2) the topological position of Carchesium and Campanella suggested that Carchesium should be placed in the family Zoothamniidae, or be elevated to a higher taxonomic rank, and that Campanella should be independent of the family Epistylididae, and probably be given a new rank; and 3) Opisthonecta grouped strongly with Astylozoon, which suggested that Opisthonecta species were not the ancestors of the stalked peritrichs.

The main goal of this study was to compare the effectiveness of three staining techniques (calcofluor white M2R, Giemsa and modified trichrome), and the polymerase chain reaction (PCR) in detecting the microsporidium Thelohania solenopsae in red imported fire ants (Solenopsis invicta). The effect of the number of ants in a sample on the sensitivity of the staining techniques and the PCR, and the effect of three DNA extraction protocols on the sensitivity of PCR were also examined. In the first protocol, the ants were macerated and the crude homogenate was used immediately in the PCR. In the second protocol, the homogenate was placed on a special membrane (FTA card) that traps DNA, which is subsequently used in the PCR. In the third protocol, the DNA was purified from the homogenate by traditional phenol-chloroform extraction. Except for PCR using FTA cards, the sensitivity (number of samples positive for T. solenopsae) of all detection techniques increased with the number of ants in the sample. Overall, Giemsa was the least sensitive of all detection techniques. Calcofluor was more sensitive than modified trichrome with ants from one site and was equally as sensitive as PCR with crude DNA or a FTA card with ants from both sites. Trichrome staining was equally as sensitive as PCR with a FTA card at both sites, but it was less sensitive than PCR with crude DNA at one site. PCR on FTA cards was less sensitive than PCR with crude DNA for ants from one site but not the other. There was no difference whether crude or phenol-chloroform purified DNA was used as template.

In summary, the results of this study show that PCR based on a crude DNA solution is equal to or more sensitive in detecting T. solenopsae than the other detection techniques investigated, and that it can be used as a reliable diagnostic tool for screening field samples of S. invicta for T. solenopsae. Nevertheless, ant smear stained with calcofluor or modified trichrome should be used to buttress findings from PCR.

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 °C and two (T5) at 42 °C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.

The cortical protein Epc1p is the most abundant protein in the membrane skeleton, or epiplasm, of Tetrahymena thermophila. A partial sequence of the EPC1 gene was obtained and used to obtain a knockout construct that was successful in transforming Tetrahymena thermophila cells. The results support the conclusion that Epc1p influences cell shape and the fidelity of cortical development. It was further observed that this protein is transferred from plus to minus cells during conjugation, and that the imported protein is assembled into the epiplasm of the recipient cell in a discreet series of steps.

Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.

A new species of Nematopsis (Apicomplexa, Porosporidae) is described from the mantle tissues of the seawater gastropod, Nerita ascencionis (Neritidae), collected in the Atlantic North off the coast of “Fernando de Noronha” Island (3° 47′ 57″ S, 32° 25′ 12″ W) situated about 350 km from the northeast coast of Brazil. Numerous oocysts, each contained in a parasitophorous vacuole, were found in the cytoplasm of phagocytes in the mantle tissue of the host. The phagocytes were surrounded by a thin wall composed of lucent material. The phagocyte cytoplasm contained a nucleus surrounded by numerous vesicles and some dense masses. The oocysts were 21.9 ± 0.5 μm long, and 11.5 ± 0.6 μm wide. The oocyst wall was 0.18–0.25 μm thick, and the apical zone contained a micropyle, 1.0–1.2 μm in diameter, covered by a canopy-like operculum about 0.25 μm thick. Externally, the oocyst wall was surrounded by numerous anastomosing microfibrils attached to the wall and extending towards the periphery of the parasitophorous vacuole. Some microfibrils formed a dense complex network that surrounded the oocyst in the middle of the parasitophorous vacuole, which opened only at the apical zone near the external region of the opercular system.

 On the basis of the data obtained by light and transmission electron microscopy and host specificity, the gregarine Nematopsis gigas is distinguished from the nearest species as a new species. The taxonomic affinities and morphological comparisons with other similar species of the same genus are discussed.

Giardiasis is the most common human protozoal infection. In their cystic phase, giardias are protected from the environment by a filamentous cyst wall made up of carbohydrates, proteins, and by two outer membranes separated from the plasma membrane of the parasite by a peripheral space. The present transmission electron microscope observations of G. lamblia cysts of human origin suggest that the extracellular peritrophic space originates from the growth, elongation, and fusion of large cytoplasmic vacuoles. As the large clear vacuoles grew in size, flattening against the inner face of the plasma membrane, they formed a single vacuole that surrounded the body of the parasite, eventually forming two outer membranes. In mature Giardia cysts, the original plasma membrane of the trophozoite becomes the outermost membrane of the cyst wall (CM1). The large vacuoles form a second membrane surrounding the cyst (CM2), and also form a third membrane (CM3), that becomes the new plasma membrane of the trophozoite. During excystation CM1 and CM2 attach to each other and fragment, leaving abundant membrane residues in the peritrophic space. Knowledge of the biochemical composition and functional properties of the complex outer membranous system of G. lamblia cysts here described will be of use to understand the survival of Giardia cysts in the environment, a major factor responsible for the high prevalence of giardiasis worldwide.

We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.

Perkinsus species are parasitic protozoa of mollusks, currently classified within the Perkinsozoa, a recently established phylum that is basal to the Apicomplexa and Dinozoa. Ribosomal RNA (rRNA) genes and their intergenic spacers have been used to support the taxonomy of Perkinsus species, the description of new species, and to develop molecular probes for their detection and identification. We previously described ultrastructure, behavior in culture, and partial sequence of the rRNA locus of a Perkinsus species isolated from the baltic clam Macoma balthica. The rRNA genes and intergenic spacers of this Perkinsus isolate differed from those described in the currently accepted species to a degree that led to its designation as a new species, Perkinsus andrewsi. In this study, we identify an additional rRNA gene unit (rRNA-B) in the P. andrewsi holotype, and report the complete sequences of both rRNA gene units. Except for the 5.8S, all regions of the rRNA-B gene unit exhibited sequence differences from that initially described (rRNA-A). Each rRNA gene unit is arranged in a “head-to-tail” tandem repeat. This is the first report demonstrating two distinct rRNA units in a Perkinsus species.

Ellobiopsids are multinucleate protist parasites of aquatic crustaceans that possess a nutrient absorbing ‘root’ inside the host and reproductive structures that protrude through the carapace. Ellobiopsids have variously been affiliated with fungi, ‘colorless algae’, and dinoflagellates, although no morphological character has been identified that definitively allies them with any particular eukaryotic lineage. The arrangement of the trailing and circumferential flagella of the rarely observed bi-flagellated ‘zoospore’ is reminiscent of dinoflagellate flagellation, but a well-organized ‘dinokaryotic nucleus' has never been observed. Using small subunit ribosomal RNA gene sequences from two species of Thalassomyces, phylogenetic analyses robustly place these ellobiopsid species among the alveolates (ciliates, apicomplexans, dinoflagellates and relatives) though without a clear affiliation to any established alveolate lineage. Our trees demonstrate that Thalassomyces fall within a dinoflagellate apicomplexa Perkinsidae “marine alveolate group 1” clade, clustering most closely with dinoflagellates. However, the poor statistical support for branches within this region indicates that additional data will be needed to resolve relationships among these taxa.

We have taken advantage of the size of the macrostomal oral apparatus of Tetrahymena vorax to investigate the immunofluorescent localization of three cytoskeletal proteins—tetrin, actin, and centrin. Tetrin and actin antibodies co-localize to cross-connectives that anchor the membranelles. These antibodies also recognize the coarse filamentous reticulum, a filament associated with the undulating membrane. Actin-specific localization extends beyond the coarse filamentous reticulum-undulating membrane complex into a region called the specialized cytoplasm. A centrin antibody localizes to the fine filamentous reticulum which, along with microtubules of the oral ribs, circumscribes the cytostomal opening. Models of phagocytic contraction based on these data are presented.