Isolation and identification of herbivorous ciliates from contaminated microalgal cultures.

Ciliates are a common but understudied group of grazers that can invade microalgal cultures. To estimate the potential impact of ciliates on microalgal culture productivity, the identification of species that can invade these cultures is essential. Furthermore, isolation of these herbivorous ciliates allows to use them in experiments that investigate the impact of ciliate grazing on the productivity of microalgal cultures. The main aims of this study were to isolate and identify ciliates that invade cultures of the freshwater microalgae Chlorella and Chlamydomonas, and to establish a live collection of these ciliates for usage in future experiments. To this end, we optimized a method for isolating ciliates from contaminated microalgal cultures and we developed a new PCR primer set for amplifying the partial 18S rDNA of ciliates belonging to the classes Spirotrichea, Oligohymenophorea and Colpodea. As a result, we isolated 11 ciliates from microalgal enrichment cultures inoculated with non-sterile dust and various freshwater sources. Of these 11 species, 7 were found to be feeding on Chlamydomonas. Ciliate species that fed on Chlorella could not be isolated in this study. Ciliate species feeding on Chlamydomonas were identified based on a combination of morphological observations and molecular analyses of partial 18S rDNA sequences.

Eukaryotic Sequences in the 16SrRNA Metagenomic Dataset of Algal-bacterial Consortia of the White Sea Coastal Zone.

The libraries of bacterial 16SrRNA gene fragment from algal-bacterial consortia of the White Sea coastal zone are analyzed. Up to 3% of the reads have revealed to correspond to eukaryotic rRNA. They related to following main eukaryotic clades: Discoba, Stramenopiles, Ciliata, Amoebozoa, and Nucletmycea. Amoebae, especially Vermamoeba, were present in all samples. In one sample, heterolobose amoeba Paravahlkampfia was detected. These microorganisms are parasites of microalgae, which can induce significant damage to industrial cultures. However, the data on their physiology and distribution are scarce. This study provides new evidence about the diversity of herbivorous eukaryotic microorganisms in natural algal-containing consortia.

Transcriptomic profiling of Alexandrium fundyense during physical interaction with or exposure to chemical signals from the parasite Amoebophrya.

Toxic microalgae have their own pathogens, and understanding the way in which these microalgae respond to antagonistic attacks may provide information about their capacity to persist during harmful algal bloom events. Here, we compared the effects of the physical presence of the parasite Amoebophrya sp. and exposure to waterborne cues from cultures infected with this parasite, on gene expression by the toxic dinoflagellates, Alexandrium fundyense. Compared with control samples, a total of 14,882 Alexandrium genes were differentially expressed over the whole-parasite infection cycle at three different time points (0, 6 and 96 h). RNA sequencing analyses indicated that exposure to the parasite and parasitic waterborne cues produced significant changes in the expression levels of Alexandrium genes associated with specific metabolic pathways. The observed upregulation of genes associated with glycolysis, the tricarboxylic acid cycle, fatty acid ß-oxidation, oxidative phosphorylation and photosynthesis suggests that parasite infection increases the energy demand of the host. The observed upregulation of genes correlated with signal transduction indicates that Alexandrium could be sensitized by parasite attacks. This response might prime the defence of the host, as indicated by the increased expression of several genes associated with defence and stress. Our findings provide a molecular overview of the response of a dinoflagellate to parasite infection.

Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus.

Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures.

A new isolate of Amoeboaphelidium protococcarum, and Amoeboaphelidium occidentale, a new species in phylum Aphelida (Opisthosporidia).

Microalgae used in the production of biofuels represents an alternative to fossil fuels. One problem in the production of algae for biofuels is attacks by algal parasitoids that can cause population crashes when algae are cultivated in outdoor ponds (Greenwell et al. 2010). Integrated solutions are being sought to mitigate this problem, and an initial step is pest identification. We isolated an algal parasitoid from an open pond of Scenedesmus dimorphus used for biofuel production in New Mexico and examined its morphology, ultrastructure and molecular phylogeny. A phylogenetic analysis placed this organism in Aphelida as conspecific with Amoeboaphelidium protococcarum sensu Karpov et al. 2013. As a result we re-evaluated the taxonomy of Amoeboaphelidium protococcarum sensu Letcher et al. 2013 and here designate it as a new species, Amoeboaphelidium occidentale.

Control of predators in industrial scale microalgae cultures with Pulsed Electric Fields.

This work describes the utilization of Pulsed Electric Fields to control the protozoan contamination of a microalgae culture, in an industrial 2.7 m(3) microalgae photobioreactor. The contaminated culture was treated with Pulsed Electric Fields, PEF, for 6h with an average of 900 V/cm, 65 µs pulses of 50 Hz. Working with recirculation, all the culture was uniformly exposed to the PEF throughout the assay. The development of the microalgae and protozoan populations was followed and the results showed that PEF is effective on the selective elimination of protozoa from microalgae cultures, inflicting on the protozoa growth halt, death or cell rupture, without affecting microalgae productivity. Specifically, the results show a reduction of the active protozoan population of 87% after 6h treatment and 100% after few days of normal cultivation regime. At the same time, microalgae growth rate remained unaffected.

Algae as reservoirs for coral pathogens.

Benthic algae are associated with coral death in the form of stress and disease. It's been proposed that they release exudates, which facilitate invasion of potentially pathogenic microbes at the coral-algal interface, resulting in coral disease. However, the original source of these pathogens remains unknown. This study examined the ability of benthic algae to act as reservoirs of coral pathogens by characterizing surface associated microbes associated with major Caribbean and Indo-Pacific algal species/types and by comparing them to potential pathogens of two dominant coral diseases: White Syndrome (WS) in the Indo-Pacific and Yellow Band Disease (YBD) in the Caribbean. Coral and algal sampling was conducted simultaneously at the same sites to avoid spatial effects. Potential pathogens were defined as those absent or rare in healthy corals, increasing in abundance in healthy tissues adjacent to a disease lesion, and dominant in disease lesions. Potentially pathogenic bacteria were detected in both WS and YBD and were also present within the majority of algal species/types (54 and 100% for WS and YBD respectively). Pathogenic ciliates were associated only with WS and not YBD lesions and these were also present in 36% of the Indo-Pacific algal species. Although potential pathogens were associated with many algal species, their presence was inconsistent among replicate algal samples and detection rates were relatively low, suggestive of low density and occurrence. At the community level, coral-associated microbes irrespective of the health of their host differed from algal-associated microbes, supporting that algae and corals have distinctive microbial communities associated with their tissue. We conclude that benthic algae are common reservoirs for a variety of different potential coral pathogens. However, algal-associated microbes alone are unlikely to cause coral death. Initial damage or stress to the coral via other competitive mechanisms is most likely a prerequisite to potential transmission of these pathogens.

Parvilucifera sinerae (Alveolata, Myzozoa) is a generalist parasitoid of dinoflagellates.

This study begins with a description of the infective process in the dinoflagellate type host Alexandrium minutum by a strain of the parasitoid, Parvilucifera sinerae, including the morphologies of the various dinoflagellate and parasitoid stages during the infection. Then, the susceptibility of 433 microalgal strains to P. sinerae infection was studied. The parasitoid was found to be capable of infecting several dinoflagellate species of the genera Alexandrium, Coolia, Dinophysis, Fragilidium, Gambierdiscus, Gymnodinium, Gyrodinium, Heterocapsa, Kryptoperidinium, Lepidodinium, Ostreopsis, Pentapharsodinium, Protoceratium, Scrippsiella, and Woloszynskia. Intra-strain variability was observed as well, such that within the same dinoflagellate species some strains were infected whereas others were not. Likewise, species of other dinoflagellate genera were not infected, such as Akashiwo, Amphidinium, Barrufeta, Bysmatrum, Karenia, Karlodinium, Prorocentrum, and Takayama. Moreover, P. sinerae was not able to infect any of the tested haptophyte, diatom, and chlorophyte species. In natural samples screened for P. sinerae infectivity, several dinoflagellate species of the genera Alexandrium, Coolia, Gonyaulax, Gymnodinium, Phalacroma, Protoperidinium, and Scrippsiella were identified as susceptible. Sporangia size was found to be proportional to the size of the host, and variations in the sporangia size were observed to influence their maturation time.

Obligately phagotrophic aphelids turned out to branch with the earliest-diverging fungi.

Reconstructing the early evolution of fungi and metazoans, two of the kingdoms of multicellular eukaryotes thriving on earth, is a challenging task for biologists. Among extant organisms having characters intermediate between fungi and hypothetical protistan ancestors, from which both fungi and metazoans are believed to have evolved, aphelids are unfairly neglected. The phylogenetic position of these microalgal endoparasites remained uncertain, since no nucleotide sequence data have been reported to date. Aphelids resemble some primitive zoosporic fungi in life cycle, but, unlike fungi, they live by phagotrophy. Here we present a phylogeny, in which a cultured aphelid species, Amoeboaphelidium protococcarum, forms a monophyletic group with Rozella and microsporidia as a sister group to Fungi. We also report a non-canonical nuclear genetic code in A. protococcarum.