Cryptic and ubiquitous aplastidic cryptophytes are key freshwater flagellated bacterivores.

Morphology-based microscopic approaches are insufficient for a taxonomic classification of bacterivorous heterotrophic nanoflagellates (HNF) in aquatic environments since their cells do not display reliably distinguishable morphological features. This leads to a considerable lack of ecological insights into this large and taxonomically diverse functional guild. Here, we present a combination of fluorescence in situ hybridization followed by catalyzed reporter deposition (CARD-FISH) and environmental sequence analyses which revealed that morphologically indistinguishable, so far largely cryptic and uncultured aplastidic cryptophytes are ubiquitous and prominent protistan bacterivores in diverse freshwater ecosystems. Using a general probe for Cryptophyceae and its heterotrophic CRY1 lineage, we analyzed different water layers in 24 freshwater lakes spanning a broad range of trophic states, sizes and geographical locations. We show that bacterivorous aplastidic cryptophytes and the CRY1 lineage accounted for ca. 2/3 and » of total HNF, respectively, in both epilimnetic and hypolimnetic samples. These heterotrophic cryptophytes were generally smaller and more abundant than their chloroplast-bearing counterparts. They had high uptake rates of bacteria, hinting at their important roles in channeling carbon flow from prokaryotes to higher trophic levels. The worldwide ubiquity of Cryptophyceae and its CRY1 lineage was supported by 18S rRNA gene sequence analyses across a diverse set of 297 freshwater metagenomes. While cryptophytes have been considered to be mainly plastidic "algae", we show that it is the aplastidic counterparts that contribute considerably to bacterial mortality rates. Additionally, our results suggest an undiscovered diversity hidden amongst these abundant and morphologically diverse aplastidic cryptophytes.

Cryptophyta as major bacterivores in freshwater summer plankton.

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator-prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Rímov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

Mixotrophy in the marine red-tide cryptophyte Teleaulax amphioxeia and ingestion and grazing impact of cryptophytes on natural populations of bacteria in Korean coastal waters.

Cryptophytes are ubiquitous and one of the major phototrophic components in marine plankton communities. They often cause red tides in the waters of many countries. Understanding the bloom dynamics of cryptophytes is, therefore, of great importance. A critical step in this understanding is unveiling their trophic modes. Prior to this study, several freshwater cryptophyte species and marine Cryptomonas sp. and Geminifera cryophila were revealed to be mixotrophic. The trophic mode of the common marine cryptophyte species, Teleaulax amphioxeia has not been investigated yet. Thus, to explore the mixotrophic ability of T. amphioxeia by assessing the types of prey species that this species is able to feed on, the protoplasms of T. amphioxeia cells were carefully examined under an epifluorescence microscope and a transmission electron microscope after adding each of the diverse prey species. Furthermore, T. amphioxeia ingestion rates heterotrophic bacteria and the cyanobacterium Synechococcus sp. were measured as a function of prey concentration. Moreover, the feeding of natural populations of cryptophytes on natural populations of heterotrophic bacteria was assessed in Masan Bay in April 2006. This study reported for the first time, to our knowledge, that T. amphioxeia is a mixotrophic species. Among the prey organisms offered, T. amphioxeia fed only on heterotrophic bacteria and Synechococcus sp. The ingestion rates of T. amphioxeia on heterotrophic bacteria or Synechococcus sp. rapidly increased with increasing prey concentrations up to 8.6×106 cells ml-1, but slowly at higher prey concentrations. The maximum ingestion rates of T. amphioxeia on heterotrophic bacteria and Synechococcus sp. reached 0.7 and 0.3 cells predator-1 h-1, respectively. During the field experiments, the ingestion rates and grazing coefficients of cryptophytes on natural populations of heterotrophic bacteria were 0.3-8.3 cells predator-1h-1 and 0.012-0.033d-1, respectively. Marine cryptophytes, including T. amphioxeia, are known to be favorite prey species for many mixotrophic and heterotrophic dinoflagellates and ciliates. Cryptophytes, therefore, likely play important roles in marine food webs and may exert a considerable potential grazing impact on the populations of marine bacteria.

Marivita cryptomonadis gen. nov., sp. nov. and Marivita litorea sp. nov., of the family Rhodobacteraceae, isolated from marine habitats.

Two strictly aerobic, Gram-negative, rod-shaped bacteria containing photosynthesis-related genes, designated strains CL-SK44(T) and CL-JM1(T), were isolated from a culture of the marine phytoplankton Cryptomonas sp. and coastal seawater from Korea, respectively. Phylogenetic analysis of 16S rRNA gene sequences revealed that the two strains were related to members of the genera Thalassobius (95.3-96.7 % similarity), Pelagibaca (95.3-96.0 %) and Donghicola (95.6 %) in the family Rhodobacteraceae. However, the two novel strains did not form a robust clade with any species of the Roseobacter clade, forming a distinct clade. The major polar lipids of the strains were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unidentified aminolipid and an unidentified lipid, profiles that were distinguishable from those of the related genera examined. Although the level of 16S rRNA gene sequence similarity between strains CL-SK44(T) and CL-JM1(T) was very high (99.1 %), DNA-DNA relatedness between the strains was 13 %, suggesting that they represent genomically distinct species. In addition, the two strains could be differentiated based on the presence of a minor polar lipid, on the hydrolysis of gelatin and the utilization of carbon sources. Based on the data from the present study, strains CL-SK44(T) and CL-JM1(T) are considered to represent separate novel species of a new genus of the family Rhodobacteraceae, for which the names Marivita cryptomonadis gen. nov., sp. nov. (type species) and Marivita litorea sp. nov. are proposed. The type strains of Marivita cryptomonadis and Marivita litorea are CL-SK44(T) (=KCCM 90070(T)=JCM 15447(T)) and CL-JM1(T) (=KCCM 90071(T)=JCM 15446(T)), respectively.