Catalyzed reported deposition-fluorescence in situ hybridization protocol to evaluate phagotrophy in mixotrophic protists

We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.     

Unveiling in situ interactions between marine protists and bacteria through single cell sequencing

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator–prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.     

Diversity and plastid types in Dinophysis acuminata complex (Dinophyceae) in Scottish waters

Dinophysis acuminata produces lipophilic shellfish toxins (LSTs) that have economic and ecological impact on marine invertebrates in NE Atlantic where aquaculture farming is prevalent. Identification of D. acuminata can be complex. Cells exhibit a variety of morphotypes that overlap between species making identification using routine light microscopy difficult. These cells are mixotrophic and their population size is influenced by hydrographic conditions and prey populations. Dinophysis cells are able to acquire and temporarily keep prey plastids from a variety of photosynthetic unicellular sources. The Dinophysis community in Scottish waters tend to be dominated by cells with morphologies that appear to be variants of D. acuminata/norvegica complex particularly during late spring/early summer. To determine the identity of these morphotypes, DNA barcoding was performed on 32 single cell isolates from sites around the Scottish coast using the ribosomal internal transcribed spacer 1 (ITS1) and a partial cytochrome oxidase I (COI) fragment on the same single cells. Although the cells exhibited a variety of morphotypes, most were restricted to one cluster containing D. acuminata and three grouped with Dinophysis ovum. This is the first molecular confirmation of the presence of D. ovum in Scottish waters. Two isolates showed considerable divergence – one was unidentifiable from the public databases, whilst the other matched a Dinophysis cf. acuta isolate from Canada. To investigate prey plastids, molecular analysis of these Dinophysis single cells was conducted with a partial fragment of the plastid ribosomal marker (16S). Most cells harboured plastids from the cryptophyte Teleaulax – the most commonly reported plastid type, however one cell harboured a Rhodomonas/Storeatula derived plastid. This finding increases the range and variety of cryptophyte plastids found in Dinophysis and increases the range of prey-types.     

Mixotrophic Protists In Marine and Freshwater Ecosystems

ABSTRACT Some protists from both marine and freshwater environments function at more than one trophic level by combining photosynthesis and panicle ingestion. Photosynthetic algae from several taxa (most commonly chrysomonads and dinoflagellates) have been reported to ingest living prey or nonliving particles, presumably obtaining part of their carbon and/or nutrients from phagocytosis. Conversely, some ciliates and sarcodines sequester chloroplasts after ingestion of algal prey. Plastid retention or "chloroplast symbiosis" by protists was first demonstrated < 20 years ago in a benthic foraminiferan. Although chloroplasts do not divide within these mixotrophic protists, they continue to function photosynthetically and may contribute to nutrition. Sarcodines and ciliates that harbor endosymbiotic algae could be considered mixotrophic but are not covered in detail here. the role of mixotrophy in the growth of protists and the impact of their grazing on prey populations have received increasing attention. Mixotrophic protists vary in their photosynthetic and ingestion capabilities, and thus, in the relative contribution of photosynthesis and phagotrophy to their nutrition. Abundant in both marine and freshwaters, they are potentially important predators of algae and bacteria in some systems. Mixotrophy may make a stronger link between the microbial and classic planktonic food webs by increasing trophic efficiency.     

A classification of mixotrophic protists based on their behaviour

1. Mixotrophs are organisms which combine phototrophy and heterotrophy; such nutritional behaviour is widespread among protists. This ability to combine multiple modes of nutrition varies between species and is not related to their taxonomic grouping. A classification of mixotrophic protists, based on their behaviour, is proposed, dividing them into four groups.
2. Group A includes protists whose primary mode of nutrition is heterotrophy and where phototrophy is employed only when prey concentrations limit heterotrophic growth. In groups B, C and D phototrophy is the dominant mode of nutrition. In group B phagotrophy supplements growth when light is limiting, therefore ingestion of prey is inversely proportional to light intensity; in group C phagotrophy provides essential substances for growth and ingestion is proportional to light intensity; and group D includes those who have very low ingestion rates, ingesting prey only, for example, for cell maintenance during prolonged dark periods.
3. This classification is aimed towards predicting the impact of any particular mixotrophic protist on the aquatic food web, and how this impact may vary depending on the environmental conditions. A model representation of the four groups is discussed.     

A classification of mixotrophic protists based on their behaviour

1. Mixotrophs are organisms which combine phototrophy and heterotrophy; such nutritional behaviour is widespread among protists. This ability to combine multiple modes of nutrition varies between species and is not related to their taxonomic grouping. A classification of mixotrophic protists, based on their behaviour, is proposed, dividing them into four groups.
2. Group A includes protists whose primary mode of nutrition is heterotrophy and where phototrophy is employed only when prey concentrations limit heterotrophic growth. In groups B, C and D phototrophy is the dominant mode of nutrition. In group B phagotrophy supplements growth when light is limiting, therefore ingestion of prey is inversely proportional to light intensity; in group C phagotrophy provides essential substances for growth and ingestion is proportional to light intensity; and group D includes those who have very low ingestion rates, ingesting prey only, for example, for cell maintenance during prolonged dark periods.
3. This classification is aimed towards predicting the impact of any particular mixotrophic protist on the aquatic food web, and how this impact may vary depending on the environmental conditions. A model representation of the four groups is discussed.     

Some factors controlling step-wise variation of organelle number in Euglena gracilis

Both nuclear and plastid ploidy double during the first 2–3 cell generations during the phosphate-induced shift from half ploidy to full ploidy in mixotrophic Euglena. Growth conditions can affect nuclear and plastid ploidy differently; plastid ploidy is more labile than nuclear ploidy during heterotrophic growth of both wild type and mutant cells, while nuclear ploidy is more labile during photoautotrophic growth.     

Element Stoichiometry of a Mixotrophic Protist Grown Under Varying Resource Conditions

ABSTRACT. The balance of essential elements (e.g. carbon [C], nitrogen [N], and phosphorus [P]) between consumers and their resources influences not only the growth and reproduction of the consumers but also the nutrients they regenerate. Flagellate protists are significant predators of aquatic bacteria and directly influence nutrient flow to higher trophic levels and, through excretion, influence the mineral element composition of dissolved nutrients. Because the element stoichiometry of protists is poorly characterized, we varied the resource composition of the bacterium Pseudomonas fluorescens and used it to grow the mixotrophic bacterivorous flagellate Ochromonas danica. Using a mass balance approach, the element composition of O. danica was found to vary depending upon the nutrient composition of the prey and ranged between 482:36:1 and 80:12:1 (C:N:P molar). Homeostasis plots suggested that flagellate protists weakly regulate their element composition and are likely to regenerate different elements depending upon the nature of the element limiting growth of their prey.     

Contrasting Mixotrophic Lifestyles Reveal Different Ecological Niches in Two Closely Related Marine Protists

Many marine microbial eukaryotes combine photosynthetic with phagotrophic nutrition, but incomplete understanding of such mixotrophic protists, their functional diversity, and underlying physiological mechanisms limits the assessment and modeling of their roles in present and future ocean ecosystems. We developed an experimental system to study responses of mixotrophic protists to availability of living prey and light, and used it to characterize contrasting physiological strategies in two stramenopiles in the genus Ochromonas. We show that oceanic isolate CCMP1393 is an obligate mixotroph, requiring both light and prey as complementary resources. Interdependence of photosynthesis and heterotrophy in CCMP1393 comprises a significant role of mitochondrial respiration in photosynthetic electron transport. In contrast, coastal isolate CCMP2951 is a facultative mixotroph that can substitute photosynthesis by phagotrophy and hence grow purely heterotrophically in darkness. In contrast to CCMP1393, CCMP2951 also exhibits a marked photoprotection response that integrates non-photochemical quenching and mitochondrial respiration as electron sink for photosynthetically produced reducing equivalents. Facultative mixotrophs similar to CCMP2951 might be well adapted to variable environments, while obligate mixotrophs similar to CCMP1393 appear capable of resource efficient growth in oligotrophic ocean environments. Thus, the responses of these phylogenetically close protists to the availability of different resources reveals niche differentiation that influences impacts in food webs and leads to opposing carbon cycle roles.     

Diversity of protists and bacteria determines predation performance and stability

Predation influences prey diversity and productivity while it effectuates the flux and reallocation of organic nutrients into biomass at higher trophic levels. However, it is unknown how bacterivorous protists are influenced by the diversity of their bacterial prey. Using 456 microcosms, in which different bacterial mixtures with equal initial cell numbers were exposed to single or multiple predators (Tetrahymena sp., Poterioochromonas sp. and Acanthamoeba sp.), we showed that increasing prey richness enhanced production of single predators. The extent of the response depended, however, on predator identity. Bacterial prey richness had a stabilizing effect on predator performance in that it reduced variability in predator production. Further, prey richness tended to enhance predator evenness in the predation experiment including all three protists predators (multiple predation experiment). However, we also observed a negative relationship between prey richness and predator production in multiple predation experiments. Mathematical analysis of potential ecological mechanisms of positive predator diversity—functioning relationships revealed predator complementarity as a factor responsible for both enhanced predator production and prey reduction. We suggest that the diversity at both trophic levels interactively determines protistan performance and might have implications in microbial ecosystem processes and services.     

Can cryptophyte abundance trigger toxic Karlodinium veneficum blooms in eutrophic estuaries?

Karlodinium veneficum is a common member of the phytoplankton in coastal ecosystems, usually present at relatively low cell abundance (10² to 10³ mL⁻¹), but capable of forming blooms of 10⁴ to 10⁵ cells mL⁻¹ under appropriate conditions. We present evidence consistent with the hypothesis that prey abundance, particularly the abundance of nano-planktonic cryptophytes, is a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments. K. veneficum is known to increase growth rate 2- to 3-fold in culture through mixotrophic nutrition, but the role of feeding in bloom formation has not been directly examined. We find that toxic K. veneficum blooms are correlated with cryptophytes abundance changes. We find a wide range of mixotrophic feeding capabilities (0–4 prey per predator per day) among genetically distinct strains of K. veneficum when fed a common prey. Finally, we find that toxic K. veneficum is capable of feeding on a wide range of cryptophyte species varying in size (31–421 μm³ per cell) and phylogenetic affinity, although ingestion rates of different prey vary significantly. While abiotic conditions (e.g. nutrients and advection) are an important aspect of K. veneficum bloom formation in eutrophic environments, our results reinforce the need for a broader view of conditions leading to toxic K. veneficum blooms including biotic factors such as prey availability.     

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter ¹. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms ².Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling ³ and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms ^{4, 2}. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web ⁵. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism ^{6, 7}. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited ^{8, 4, 9, 10, 5}. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity ^{4, 11}, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs ¹². In this study, we applied a radioisotope assay modified for filtered samples ¹³ to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.     

Interactions between the mixotrophic dinoflagellate Takayama helix and common heterotrophic protists

The phototrophic dinoflagellate Takayama helix that is known to be harmful to abalone larvae has recently been revealed to be mixotrophic. Although mixotrophy elevates the growth rate of T. helix by 79%–185%, its absolute growth rate is still as low as 0.3 d⁻¹. Thus, if the mortality rate of T. helix due to predation is high, this dinoflagellate may not easily prevail. To investigate potential effective protistan grazers on T. helix, feeding by diverse heterotrophic dinoflagellates such as engulfment-feeding Oxyrrhis marina, Gyrodinium dominans, Gyrodinium moestrupii, Polykrikos kofoidii, and Noctiluca scintillans, peduncle-feeding Aduncodinium glandula, Gyrodiniellum shiwhaense, Luciella masanensis, and Pfiesteria piscicida, pallium-feeding Oblea rotunda and Protoperidinium pellucidum, and the naked ciliates Pelagostrobilidium sp. (ca. 40 μm in cell length) and Strombidinopsis sp. (ca. 150 μm in cell length) on T. helix was explored. Among the tested heterotrophic protists, O. marina, G. dominans, G. moestrupii, A. glandula, L. masanensis, P. kofoidii, P. piscicida, and Strombidinopsis sp. were able to feed on T. helix. The growth rates of all these predators except Strombidinopsis sp. with T. helix prey were lower than those without the prey. The growth rate of Strombidinopsis sp. on T. helix was almost zero although the growth rate of Strombidinopsis sp. with T. helix prey was higher than those without the prey. Moreover, T. helix fed on O. marina and P. pellucidum and lysed the cells of P. kofoidii and G. shiwhaense. With increasing the concentrations of T. helix, the growth rates of O. marina and P. kofoidii decreased, but those of G. dominans and L. masanensis largely did not change. Therefore, reciprocal predation, lysis, no feeding, and the low ingestion rates of the common protists preying on T. helix may result in a low mortality rate due to predation, thereby compensating for this species’ low growth rate.     

Mixotrophic trade‐off under warming and UVR in a marine and a freshwater alga

Mixotrophic protists combine phagotrophy and phototrophy within a single cell. Greater phagotrophic activity could reinforce the bypass of carbon (C) flux through the bacteria‐mixotroph link and thus lead to a more efficient transfer of C and other nutrients to the top of the trophic web. Determining how foreseeable changes in temperature and UVR affect mixotrophic trade‐offs in favor of one or the other nutritional strategy, along the mixotrophic gradient, is key to understanding the fate of carbon and mineral nutrients in the aquatic ecosystem. Our two main hypotheses were: (i) that increased warming and UVR will divert metabolism toward phagotrophy, and (ii) that the magnitude of this shift will vary according to the organism's position along the mixotrophic gradient. To test these hypotheses, we used two protists (Isochrysis galbana and Chromulina sp.) located in different positions on the mixotrophic gradient, subjecting them to the action of temperature and of UVR and their interaction. Our results showed that the joint action of these two factors increased the primary production:bacterivory ratio and stoichiometric values (N:P ratio) close to Redfield's ratio. Therefore, temperature and UVR shifted the metabolism of both organisms toward greater phototrophy regardless of the original position of the organism on the mixotrophic gradient. Weaker phagotrophic activity could cause a less efficient transfer of C to the top of trophic webs.     

On the morphology and predatory behavior of the dinoflagellate Oxyrrhis marina exposed to reduced salinity

Changes in salinity are known to alter the morphology of protists, and we hypothesized that these changes subsequently alter also the predatory behavior of the dinoflagellate Oxyrrhis marina. Oxyrrhis was grown in media of 33, 25, 20, and 10% of the regular salinity of f/2 medium (31–32‰). In all cases, the cells discharged trichocysts and swelled. Cell surfaces and volumes increased with decreasing salinity, such that cell surface area at least doubled at 10% and the cell volume increased approximately fourfold. After 1 h, the cells started to regain their regular shape, which was almost completed after 24 h. Oxyrrhis immediately regained its regular shape when culture medium was added 5–10 min after the osmotic stress. When incubated with Pyramimonas grossii as prey, those short-term stressed cells showed no significant different prey uptake in comparison to non-stressed cells. In contrast, 24 h after the addition of prey, short-term stressed Oxyrrhis cells had, with weak statistical significance, more Pyramimonas cells engulfed than non-stressed cells. These results indicated that (1) trichocysts were most likely not involved in prey capture and (2) salinity-stressed Oxyrrhis either enhanced its capability to capture more prey, or its digestion apparatus was hampered.     

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⁻¹ h⁻¹ and 0.012–0.033 d⁻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.     

CYANOPHYTE AND CYANELLE DNA: A SEARCH FOR THE ORIGINS OF PLASTIDS1

Cyanophyte-like prokaryotes are widely presumed to be the progenitors of eukaryote plastids. A few rare protistan species bearing cyanophyte-like cyanelles may represent intermediate stages in the evolution of true organelles. Cyanophyte DNA disposition in the cell, so far as is known from electron microscopy, seems uniform within the group and distinctly different from the several known arrangements of DNA in plastids. Therefore a survey of representative cyanophytes and protistan cyanelles was undertaken to determine whether forms reminiscent of plastids could be found. DNA-specific fluorochromes were utilized, along with epifluorescent microscopy, to study the DNA arrangement in situ in whole cells. Only the endospore (baeocyte)-forming Cyanophyta contained more than one, centrally located DNA skein per cell, and then only for the period just preceding visible baeocyte formation. Such forms might, with modification, presage the “scattered nucleoid” DNA disposition found in plastids of several groups, including Rhodophytes, Cryptophytes, Chlorophytes and higher plants. The DNA arrangement in cyanelles of two protists, Cyanophora and Glaucocystis, appear different from each other and possibly related to, respectively, the cyanophytes Gloeobacter and Synechococcus. Cyanelles of the third protist, Glaucosphaera, like the cells of the unique prokaryote Prochloron, appear to have multiple sites of DNA, somewhat similar to those of the “scattered nucleoid” line of plastid evolution. No obvious precursor of the “ring nucleoid” or other types of plastid DNA conformation was found.     

Bacterial resistance to arsenic protects against protist killing

Protists kill their bacterial prey using toxic metals such as copper. Here we hypothesize that the metalloid arsenic has a similar role. To test this hypothesis, we examined intracellular survival of Escherichia coli (E. coli) in the amoeba Dictyostelium discoideum (D. discoideum). Deletion of the E. coli ars operon led to significantly lower intracellular survival compared to wild type E. coli. This suggests that protists use arsenic to poison bacterial cells in the phagosome, similar to their use of copper. In response to copper and arsenic poisoning by protists, there is selection for acquisition of arsenic and copper resistance genes in the bacterial prey to avoid killing. In agreement with this hypothesis, both copper and arsenic resistance determinants are widespread in many bacterial taxa and environments, and they are often found together on plasmids. A role for heavy metals and arsenic in the ancient predator–prey relationship between protists and bacteria could explain the widespread presence of metal resistance determinants in pristine environments.     

In situ plastid and mitochondrial DNA determination: Implication of the observed MINIMAL PLASTID GENOME NUMBER

The total loss of plastid DNA has never been reported for any alga or plant cell line, with the sole exception of the protozoan Euglena, yet plastid distribution at mitosis is apparently stochastric (Birky and Skavaril, Journal of Theoretical Biology, vol. 106, pp. 441–447, 1984) and accidental loss might be expected. It is not obvious how stem cells of photosynthetic eukaryotes avoid this problem. The chrysophyte alga Ochromonas danica, described as having but one or two plastids, can proliferate indefinitely without the benefit of photosynthesis. Under such conditions its plastid genome copy number per cell might drop to the absolute minimum compatible with maintaining its inheritance. In situ quantitation of Ochromonas plastid DNA in both photosynthetic and enriched mixotrophic growth, and in heterotrophic growth in prolonged darkness, suggests that plastids are capable of very wide variation (7 to >;200 genomes/plastid) in their DNA content, and likewise, cells can vary from one to >;8 plastids per cell, with total genomes numbers from 7 to >;1,000 per cell. Among many growth conditions tested, the smallest plastids were found in rapidly dividing cells grown in the dark, many of which contained but one plastid. The inability to find plastids with fewer than seven plastid genome equivalents of DNA, even in these rapidly multiplying cells grown in total darkness for months, suggests that multiple copies of the plastid genome may be very carefully maintained, even in the prolonged absence of photosynthesis. This implies that multiple copies are important for reasons other than photosynthetic capability; two possibilities are the biosynthetic steps necessary for eukaryote cell survival known to occur solely within a plastid, and/or the potential that multiple plastid genome copies provide to escape the effects of Muller's ratchet.     

Holotranscobalamins in B12 and non B12 requiring prokaryotes and eukaryotes

Transcobalamins, vitamin B₁₂ binding proteins, deliver B₁₂ to cell surface receptors which then permit B₁₂ to cross cell membranes for metabolic use. There is little documentation concerning B₁₂ binding proteins in bacteria and protists. We found that prokaryotes and eukaryotes requiring B₁₂, as well as those protists synthesizing B₁₂, also produce several transcobalamins for functionally transporting B₁₂ similar to humans.