Environment-dependent metabolic investments in the mixotrophic chrysophyte Ochromonas.

Mixotrophic protists combine photosynthesis and phagotrophy to obtain energy and nutrients. Because mixotrophs can act as either primary producers or consumers, they have a complex role in marine food webs and biogeochemical cycles. Many mixotrophs are also phenotypically plastic and can adjust their metabolic investments in response to resource availability. Thus, a single species's ecological role may vary with environmental conditions. Here, we quantified how light and food availability impacted the growth rates, energy acquisition rates, and metabolic investment strategies of eight strains of the mixotrophic chrysophyte, Ochromonas. All eight Ochromonas strains photoacclimated by decreasing chlorophyll content as light intensity increased. Some strains were obligate phototrophs that required light for growth, while other strains showed stronger metabolic responses to prey availability. When prey availability was high, all eight strains exhibited accelerated growth rates and decreased their investments in both photosynthesis and phagotrophy. Photosynthesis and phagotrophy generally produced additive benefits: In low-prey environments, Ochromonas growth rates increased to maximum, light-saturated rates with increasing light but increased further with the addition of abundant bacterial prey. The additive benefits observed between photosynthesis and phagotrophy in Ochromonas suggest that the two metabolic modes provide nonsubstitutable resources, which may explain why a tradeoff between phagotrophic and phototrophic investments emerged in some but not all strains.

Functional control and metabolic integration of stolen organelles in a photosynthetic ciliate.

Stealing prey plastids for metabolic gain is a common phenomenon among protists within aquatic ecosystems.1 Ciliates of the Mesodinium rubrum species complex are unique in that they also steal a transcriptionally active but non-dividing prey nucleus, the kleptokaryon, from certain cryptophytes.2 The kleptokaryon enables full control and replication of kleptoplastids but has a half-life of about 10 days.2 Once the kleptokaryon is lost, the ciliate experiences a slow loss of photosynthetic metabolism and eventually death.2,3,4 This transient ability to function phototrophically allows M. rubrum to form productive blooms in coastal waters.5,6,7,8 Here, we show, using multi-omics approaches, that an Antarctic strain of the ciliate not only depends on stolen Geminigera cryophila organelles for photosynthesis but also for anabolic synthesis of fatty acids, amino acids, and other essential macromolecules. Transcription of diverse pathways was higher in the kleptokaryon than that in G. cryophila, and many increased in higher light. Proteins of major biosynthetic pathways were found in greater numbers in the kleptokaryon relative to M. rubrum, implying anabolic dependency on foreign metabolism. We show that despite losing transcriptional control of the kleptokaryon, M. rubrum regulates kleptoplastid pigments with changing light, implying an important role for post-transcriptional control. These findings demonstrate that the integration of foreign organelles and their gene and protein expression, energy metabolism, and anabolism occur in the absence of a stable endosymbiotic association. Our results shed light on potential events early in the process of complex plastid acquisition and broaden our understanding of symbiogenesis.

Cascading effects of prey identity on gene expression in a kleptoplastidic ciliate.

Kleptoplastidic, or chloroplast stealing, lineages transiently retain functional photosynthetic machinery from algal prey. This machinery, and its photosynthetic outputs, must be integrated into the host's metabolism, but the details of this integration are poorly understood. Here, we study this metabolic integration in the ciliate Mesodinium chamaeleon, a coastal marine species capable of retaining chloroplasts from at least six distinct genera of cryptophyte algae. To assess the effects of feeding history on ciliate physiology and gene expression, we acclimated M. chamaeleon to four different types of prey and contrasted well-fed and starved treatments. Consistent with previous physiological work on the ciliate, we found that starved ciliates had lower chlorophyll content, photosynthetic rates, and growth rates than their well-fed counterparts. However, ciliate gene expression mirrored prey phylogenetic relationships rather than physiological status, suggesting that, even as M. chamaeleon cells were starved of prey, their overarching regulatory systems remained tuned to the prey type to which they had been acclimated. Collectively, our results indicate a surprising degree of prey-specific host transcriptional adjustments, implying varied integration of prey metabolic potential into many aspects of ciliate physiology.

Evidence for evolutionary adaptation of mixotrophic nanoflagellates to warmer temperatures.

Mixotrophs, organisms that combine photosynthesis and heterotrophy to gain energy, play an important role in global biogeochemical cycles. Metabolic theory predicts that mixotrophs will become more heterotrophic with rising temperatures, potentially creating a positive feedback loop that accelerates carbon dioxide accumulation in the atmosphere. Studies testing this theory have focused on phenotypically plastic (short-term, non-evolutionary) thermal responses of mixotrophs. However, as small organisms with short generation times and large population sizes, mixotrophs may rapidly evolve in response to climate change. Here, we present data from a 3-year experiment quantifying the evolutionary response of two mixotrophic nanoflagellates to temperature. We found evidence for adaptive evolution (increased growth rates in evolved relative to acclimated lineages) in the obligately phototrophic strain, but not in the facultative phototroph. All lineages showed trends of increased carbon use efficiency, flattening of thermal reaction norms, and a return to homeostatic gene expression. Generally, mixotrophs evolved reduced photosynthesis and higher grazing with increased temperatures, suggesting that evolution may act to exacerbate mixotrophs' effects on global carbon cycling.

Codependence of individuals in the Nephromyces species swarm requires heterospecific bacterial endosymbionts.

Symbiosis is one of the most important evolutionary processes shaping the biodiversity on Earth. Symbiotic associations often bring together organisms from different domains of life, which can provide an unparalleled route to evolutionary innovation.1-4 The phylum Apicomplexa encompasses 6,000 ubiquitous animal parasites; however, species in the recently described apicomplexan family, Nephromycidae, are reportedly non-virulent.5,6 The members of the genus Nephromyces live within a specialized organ of tunicates, called the renal sac, in which they use concentrated uric acid as a primary nitrogen source.7,8 Here, we report genomic and transcriptomic data from the diverse genus Nephromyces, as well as the three bacterial symbionts that live within this species complex. We show that the diversity of Nephromyces is unexpectedly high within each renal sac, with as many as 20 different species inhabiting the renal sacs in wild populations. The many species of Nephromyces can host three different types of bacterial endosymbionts; however, FISH microscopy allowed us to demonstrate that each individual Nephromyces cell hosts only a single bacterial type. Through the reconstruction and analyses of the endosymbiont bacterial genomes, we infer that each bacterial type supplies its host with different metabolites. No individual species of Nephromyces, in combination with its endosymbiont, can produce a complete set of essential amino acids, and culture experiments demonstrate that individual Nephromyces species cannot form a viable infection. Therefore, we hypothesize that Nephromyces spp. depend on co-infection with congeners containing different bacterial symbionts in order to exchange metabolites to meet their needs.

Metabolic Contributions of an Alphaproteobacterial Endosymbiont in the Apicomplexan Cardiosporidium cionae.

Apicomplexa is a diverse protistan phylum composed almost exclusively of metazoan-infecting parasites, including the causative agents of malaria, cryptosporidiosis, and toxoplasmosis. A single apicomplexan genus, Nephromyces, was described in 2010 as a mutualist partner to its tunicate host. Here we present genomic and transcriptomic data from the parasitic sister species to this mutualist, Cardiosporidium cionae, and its associated bacterial endosymbiont. Cardiosporidium cionae and Nephromyces both infect tunicate hosts, localize to similar organs within these hosts, and maintain bacterial endosymbionts. Though many other protists are known to harbor bacterial endosymbionts, these associations are completely unknown in Apicomplexa outside of the Nephromycidae clade. Our data indicate that a vertically transmitted α-proteobacteria has been retained in each lineage since Nephromyces and Cardiosporidium diverged. This α-proteobacterial endosymbiont has highly reduced metabolic capabilities, but contributes the essential amino acid lysine, and essential cofactor lipoic acid to C. cionae. This partnership likely reduces resource competition with the tunicate host. However, our data indicate that the contribution of the single α-proteobacterial endosymbiont in C. cionae is minimal compared to the three taxa of endosymbionts present in the Nephromyces system, and is a potential explanation for the virulence disparity between these lineages.

Nephromyces Represents a Diverse and Novel Lineage of the Apicomplexa That Has Retained Apicoplasts.

A most interesting exception within the parasitic Apicomplexa is Nephromyces, an extracellular, probably mutualistic, endosymbiont found living inside molgulid ascidian tunicates (i.e., sea squirts). Even though Nephromyces is now known to be an apicomplexan, many other questions about its nature remain unanswered. To gain further insights into the biology and evolutionary history of this unusual apicomplexan, we aimed to 1) find the precise phylogenetic position of Nephromyces within the Apicomplexa, 2) search for the apicoplast genome of Nephromyces, and 3) infer the major metabolic pathways in the apicoplast of Nephromyces. To do this, we sequenced a metagenome and a metatranscriptome from the molgulid renal sac, the specialized habitat where Nephromyces thrives. Our phylogenetic analyses of conserved nucleus-encoded genes robustly suggest that Nephromyces is a novel lineage sister to the Hematozoa, which comprises both the Haemosporidia (e.g., Plasmodium) and the Piroplasmida (e.g., Babesia and Theileria). Furthermore, a survey of the renal sac metagenome revealed 13 small contigs that closely resemble the genomes of the nonphotosynthetic reduced plastids, or apicoplasts, of other apicomplexans. We show that these apicoplast genomes correspond to a diverse set of most closely related but genetically divergent Nephromyces lineages that co-inhabit a single tunicate host. In addition, the apicoplast of Nephromyces appears to have retained all biosynthetic pathways inferred to have been ancestral to parasitic apicomplexans. Our results shed light on the evolutionary history of the only probably mutualistic apicomplexan known, Nephromyces, and provide context for a better understanding of its life style and intricate symbiosis.

Nephromyces Encodes a Urate Metabolism Pathway and Predicted Peroxisomes, Demonstrating That These Are Not Ancient Losses of Apicomplexans.

The phylum Apicomplexa is a quintessentially parasitic lineage, whose members infect a broad range of animals. One exception to this may be the apicomplexan genus Nephromyces, which has been described as having a mutualistic relationship with its host. Here we analyze transcriptome data from Nephromyces and its parasitic sister taxon, Cardiosporidium, revealing an ancestral purine degradation pathway thought to have been lost early in apicomplexan evolution. The predicted localization of many of the purine degradation enzymes to peroxisomes, and the in silico identification of a full set of peroxisome proteins, indicates that loss of both features in other apicomplexans occurred multiple times. The degradation of purines is thought to play a key role in the unusual relationship between Nephromyces and its host. Transcriptome data confirm previous biochemical results of a functional pathway for the utilization of uric acid as a primary nitrogen source for this unusual apicomplexan.

Preadult parental diet affects offspring development and metabolism in Drosophila melanogaster.

When Drosophila melanogaster larvae are reared on isocaloric diets differing in their amounts of protein relative to sugar, emerging adults exhibit significantly different development times and metabolic pools of protein, glycogen and trigylcerides. In the current study, we show that the influence of larval diet experienced during just one generation extends into the next generation, even when that subsequent generation had been shifted to a standard diet during development. Offspring of flies that were reared on high protein relative to sugar underwent metamorphosis significantly faster, had higher reproductive outputs, and different metabolic pool contents compared to the offspring of adults from low protein relative to sugar diets. In addition, isofemale lines differed in the degree to which parental effects were observed, suggesting a genetic component to the observed transgenerational influences.

Dietary protein and sugar differentially affect development and metabolic pools in ecologically diverse Drosophila.

We examined the effects of 3 diets differing in their relative levels of sugar and protein on development and metabolic pools (protein, TG, and glycogen) among sets of isofemale lines of 2 ecologically distinct Drosophila species, D. melanogaster and D. mojavensis. Our high protein:sugar ratio diet contained 7.1% protein and 17.9% carbohydrate, the EPS diet was 4.3% protein and 21.2% carbohydrate, and the LPS was only 2.5% protein and 24.6% carbohydrate. Larvae of D. melanogaster, a generalist fruit breeder, were able to survive on all 3 diets, although all 3 metabolic pools responded with significant diet and diet × line interactions. Development was delayed by the diet with the most sugar relative to protein. The other species, D. mojavensis, a cactus breeder ecologically unaccustomed to encountering simple sugars, completely failed to survive when fed the diet with the highest sugar and showed very poor survival even with the diet with equal parts of protein and sugar. Furthermore, the D. mojavensis adult metabolic pools of protein, TG, and glycogen significantly differed from those of D. melanogaster adults fed the identical diet. Thus, considerable within- and between-species differences exist in how diets are metabolized. Given that the genomes of both of these Drosophila species have been sequenced, these differences and their genetic underpinnings hold promise for understanding human responses to nutrition and for developing strategies for dealing with metabolic disease.