Thank you. Good photographs are required to support records. All year round — hydra do not seem to age, but may die of things like disease or starvation. Typically they feed on small crustaceans, insects and annelids. True status in Britain is difficult to determine due to lack of records. As for ammonium, which is one of the main nitrogen sources in plants, previous studies have reported the inability of symbiotic algae to take up ammonium because of the low peri-algal pH pH 4—5 that stimulates maltose release Douglas and Smith, ; Rees, ; McAuley, ; Dorling et al.
Since Chlorella apparently cannot use nitrite and ammonium as a nitrogen source, it seems that Hydra has to assimilate ammonium to glutamine and provides it to Chlorella A99 Figure 8A. A Summary of symbiotic interactions between Hydra and Chlorella A During light conditions, Chlorella A99 performs photosynthesis and produces maltose Mal , which is secreted into the Hydra symbiosome where it is possibly digested to glucose Gluc , shown in red.
Since the sugar also up-regulates the NaPi gene, which controls intracellular phosphate levels, it might be involved in the supply of phosphorus to Chlorella as well blue broken line. The sugar is transported to the ectoderm red broken line and there induces the expression of GS and Spot In the Chlorella A99 genome, degeneration of the nitrate assimilation system and an increase of amino acid transporters was observed green balloon.
Red indicates transfer of photosynthesis products from the symbiont to the host, and blue indicates transfer of nitrogen sources from the host to the symbiont.
Previous studies have reported that symbiotic Chlorella in green hydra releases significantly larger amounts of maltose than NC64A Mews and Smith, ; Rees, In addition, Rees reported that Hydra polyps containing high maltose releasing algae had a high GS activity, whereas aposymbiotic Hydra or Hydra with a low maltose releasing algae had lower GS activity Rees, Although the underlying mechanism of how maltose secretion and transportation from Chlorella is regulated is still unclear, the amount of maltose released by the symbiont could be an important symbiont-derived driver or stabilizer of the Hydra—Chlorella symbiosis.
Transcriptome comparison between symbiotic and aposymbiotic H. The fact that PRRs and apoptosis-related genes, are also differentially expressed in a number of other symbiotic cnidarians Table 1 , suggests innate immunity as conserved mechanism involved in controlling the development and maintenance of stable symbiotic interactions.
Furthermore, the exchange of nitrogenous compounds and photosynthetic products between host and symbiont observed here in the Hydra-Chlorella symbiosis is also observed in marine invertebrates such as corals, sea anemones and giant clams associated with Symbiodinium algae Figure 8B,C.
Despite these similarities, however, there are also conspicuous differences among symbiotic cnidarians in particular with respect to the nutrients provided by the symbiont to the host. For example, symbiotic Chlorella algae in green hydra, Paramecium and fresh water sponges provide their photosynthetic products in form of maltose and glucose Figure 8B Brown and Nielsen, ; Wilkinson, ; Kamako and Imamura, In contrast, Symbiodinium provides glucose, glycerol, organic acids, amino acids as well as lipids to its marine hosts Figure 8C Muscatine and Cernichiari, ; Lewis and Smith, ; Trench, ; Kellogg and Patton, A former transcriptome analysis of amino acid biosynthetic pathways suggested that Symbiodinium can synthesize almost all amino acids Shinzato et al.
Gene loss in cysteine synthesis pathway in the coral host Acropora digitifera seems to reflect the dependency on the amino acids provided by the Symbiodinium symbiont Shinzato et al. In contrast to Symbiodinium which can assimilate inorganic nitrogen such as nitrate and ammonium Lipschultz and Cook, ; Grover et al. The nitrogen conservation hypothesis suggests that photosynthetic carbon compounds from the symbiont are used preferentially by the host respiration, which reduces catabolism of nitrogenous compounds Rees and Ellard, ; Szmant et al.
Our observation that in symbiotic green hydra many genes involved in amino acid metabolism are down-regulated Figure 1E is consistent with the assumption of reduction of amino acid consumption by respiration. In addition to the nitrogen recycling hypothesis, it has been proposed that also corals, sea anemones, Paramecium and green hydra hosts can assimilate ammonium into amino acids Figure 8B,C Host nitrogen assimilation Miller and Yellowlees, ; Rees, ; Szmant et al. Ammonia assimilation by the host implies that the host controls the nitrogen status to regulate metabolism of the symbionts, which may be involved in controlling the number of symbionts within the host cell.
For organisms such as corals living in oligotrophic sea, inorganic nitrogen assimilation and recycling may be necessary to manage the nitrogen sources efficiently. In contrast, for Hydra and Paramecium living in a relatively nutrient-rich environment may be advantageous in terms of metabolic efficiency that the symbiont abandons its ability to assimilate inorganic nitrogen and specializes in the supply of photosynthetic carbohydrate to the host.
Metabolic dependence of symbionts on host supply occasionally results in genome reduction and gene loss. For example, symbiotic Buchnera bacteria in insects are missing particular genes in essential amino acid pathways Shigenobu et al.
The fact that the corresponding genes of the host are up-regulated in the bacteriocyte, indicates complementarity and syntrophy between host and symbiont.
Similarly, in Chlorella A99 the nitrogen assimilation system could have been lost as a result of continuous supply of nitrogenous amino acids provided by Hydra. Compared to Chlorella NC64A, the closest relative to Chlorella A99 among the genome-sequenced algae, genome size and total number of genes in Chlorella A99 were found to be smaller Figure 4B. NR and NiR activities were found to be induced by nitrate in free-living Chlorella , but not in Chlorella NC64A, indicating mutations in the regulatory region Kamako et al.
Considering the phylogenetic position of NC64A and the symbiotic Chlorella of green hydra Kawaida et al. By contrast, the parasitic algae Helicosporidium and Auxochlorella have significantly smaller genome sizes and number of genes indicating extensive genome reduction Gao et al.
The apparently unchanged complexity of the Chlorella A99 genome suggests a relatively early stage of this symbiotic partnership. Thus, gene loss in metabolic pathways could occur as a first step of genome reduction in symbionts caused by the adaptation to continuous nutrient supply from the host.
Taken together, our study suggests metabolic-codependency as the primary driving force in the evolution of symbiosis between Hydra and Chlorella. Experiments were carried out with the Australian Hydra viridissima strain A99, which was obtained from Dr. Richard Campbell, Irvine. Experiments were carried out with polyps starved for 3—6 days. Isolation of endodermal layer and ectodermal layer was performed as described by Kishimoto et al.
Kishimoto et al. Experiments were carried out using three biological replicates. The intensity of probes was extracted from scanned microarray images using Feature Extraction All algorithms and parameters used in this analysis were used with default conditions.
Background-subtracted signal-intensity values gProcessedSignal generated by the Feature Extraction software were normalized using the 75 th percentile signal intensity among the microarray.
Total RNA was extracted from 50 green hydra polyps for each biological replicate independently. All qPCR experiments were performed in duplicate with three biological replicates each. Values were normalized using the expression of the tubulin alpha gene. Primers used for these experiments are listed in Supplementary file 6A.
Expression patterns of specific Hydra genes were detected by whole mount in situ hybridization with digoxigenin DIG -labelled RNA probes. DIG-labeled sense probes targeting the same sequences as the antisense probes were used as a control.
Primers used for these experiments are listed in Supplementary file 6B. For genome sequencing of Chlorella sp. A99, Chlorella sp. A99 was isolated from H. Paired-end library insert size: bp and mate-pair libraries insert size: 2. Genome sequencing was performed using Illumina Miseq and Hiseq platforms. Sequence reads were assembled using Newbler Assembler version 2.
Gaps inside the scaffolds were closed with the paired-end and mate-pair data using GapCloser of Short Oligonucleotide Analysis Package Luo et al. To examine the conservation of H. For comparative analysis of gene models of Chlorella sp. A99 and other algae, domain searches against the Pfam database Pfam-A. Nitrogen assimilation genes in Chlorella A99 were identified by orthologous gene groups and reciprocal blast searches. The number of genes for nitrate assimilation genes, glutamine synthetase and glutamate synthetase, and clustering of such genes were systematically reported by Sanz-Luque et al.
We used these data as reference for searches of nitrogen assimilation genes, and further nitrogen assimilation genes were searched by Kyoto Encyclopedia of Genes and Genomes KEGG pathway Kanehisa and Goto, JGI genome browsers of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C were also used for retrieving genes and checking gene order on the scaffolds. Sequences of poor quality that did not well align were deleted using BioEdit Hall, Primers for NAR2 could not be designed because of insufficient conservation.
In each case, 10 ng gDNA was used as a template. The primers used are described in Supplementary file 6C. To isolate symbiotic algae, polyps were quickly homogenized in 0. The pellet was resuspended in 0. Sequencing was performed as described above. The primers used are described in Supplementary file 6D. All the results of microarray analysis are included in Supplementary Table 1. The Whole Genome Shotgun project of Chlorella sp. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses.
A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "Metabolic co-dependence drives the evolutionary ancient Hydra-Chlorella symbiosis" for consideration by eLife.
Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ian Baldwin as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Virginia M Weis Reviewer 1 ; Eunsoo Kim Reviewer 2.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. This manuscript describes genomic and transcriptomic evidence for the metabolic interdependence of the partners in the Hydra-Chlorella symbiosis.
The work was carefully designed and executed and it is presented in a well-constructed and well-written manuscript. The genome based investigation confirmed that the consortium is driven by nutrient exchanges: some animal genes identified in this study have implications in carbohydrate metabolism whereas the algal genome suggests its reduced competency in assimilation of inorganic source of nitrogen and an increased capacity to transport amino acids.
The work produced, among other things, a high-quality genome assembly for the symbiotic Chlorella sp. The study casts the work in an evolutionary and historical light. The researchers do a good job of capturing the excellent original green Hydra literature from the late 20th century and using it as a springboard for their modern, forward looking take on the classical questions in symbiosis. They center the story around metabolic interaction and make a good case with their data for showing metabolic interdependence, as evidenced by reduced nitrogen assimilation pathways in the symbiont, localization of symbiosis genes in the host, and light experiments showing that expression is tied to symbiont photosynthesis.
While we largely support the metabolic interaction storyline, we feel that the authors undersell some of their interesting findings — they seem to sweep them under the carpet. They show that some of the most differentially expressed genes in the host are in innate immune pathways and they even find that expression of the genes is not directly linked to symbiont productivity. Yet, the authors chose not to discuss innate immunity and its role in symbiosis at all, nor do they discuss the expression data — except to use them as almost a control for the metabolic gene expression patterns.
Hence we feel that this is a missed opportunity and is a little surprising given Bosch and colleagues' expertise in the role of innate immunity in symbiosis. Similarly, the authors don't linger on the differences in expression when hosts are colonized by an inappropriate symbiont. We view this as another missed opportunity. Another major component missing in this manuscript is a bird's eye view comparison to other relevant large-scale sequencing based studies.
As authors pointed out in the Discussion, there are examples of endosymbioses that are based on nitrogen and carbohydrate nutrients, including those, such as coral- Symbiodinium, Paramecium-Chlorella , and salamander-green alga, that have been investigated by transcriptome approach. I'd thus encourage authors to discuss as to how similar and dissimilar the major molecular mechanisms of these distinct, yet ecologically comparable associations are. Please expand the Discussion to include comparisons between your data and the much larger field of anthozoan-dinoflagellate symbiosis and other large scale genome analyses that are relevant.
While I largely support the metabolic interaction storyline, I feel that the authors undersell some of their interesting findings — they seem to sweep them under the carpet. I feel that this is a missed opportunity and is a little surprising given Bosch and colleagues' expertise in the role of innate immunity in symbiosis. I view this as another missed opportunity.
The authors have opted to keep the Discussion short and have not expanded it to include comparisons between their data and the much larger field of anthozoan-dinoflagellate symbiosis. There are many interesting parallels and differences that they could discuss — the most interesting to me being the differences in symbiont genome size the similarities for evidence loss of some pathways. Again, this is surprising given Satoh and Shinzato's expertise in coral genomics. I view this as another missed opportunity that undersells the impact of this study.
I found the naming of the organisms and the partnerships inconsistent and somewhat confusing. For example, the naming is different in Figure 1 and Table 1. Sometimes the symbiont strain name is used to represent its presence in the host — like in Table 1, but sometimes not, like in Figure 1.
The authors also need to check their terminology throughout the manuscript and strive for consistency. Sometimes the authors use photobiont, but others times just symbiont — etc. The term 'zooxanthellae' is no longer used in the literature. I would suggest changing to ' Symbiodinium '. Where is the complete dataset of differentially expressed genes summarized in Figure 1C?
Will this list be made available to the reader? There is a new paper by Sproles et al. MPE that extensively examines transporters in cnidarian- Symbiodinium symbioses and includes discussion of nitrogen transporters.
This paper should be included in their Discussion. In Figure 7, how would Gln be transported across the host symbiosome? There is no transporter depicted there. This is an interesting study that describes Hydra -green algal symbiosis based on the use of large-scale genetic data. Their genome based investigation confirmed that the consortium is driven by nutrient exchanges: some animal genes identified in this study have implication in carbohydrate metabolism whereas the algal genome suggests its reduced competency in assimilation of inorganic source of nitrogen and an increased capacity to transport amino acids.
Abstract "in an unbiased approach.. For the alga, the authors focused specifically to select nitrogen metabolism related genes based on earlier studies. Thus, it is highly likely that other molecular aspects of algal responses to symbiosis may have been overlooked. Perhaps the authors could do an "unguided" comparison of the algal genome to its related, non-symbiotic genomes. If this proves to be too daunting, I suggest minimally the authors clearly indicate in the text the limitation of their experimental approach.
While reviewing this article, I came across the paper by Ishikawa et al. In the study, authors generated and compared transcriptomes of two different species of Hydra that were grown with or without green algal symbiont.
In that RNA-seq based experiment, 1, contigs were found to be up-regulated and 2, to be down-regulated for symbiotic H. In contrast, Hamada et al. As the two studies overlap quite a bit, it seems important that authors compare their results to the study data and address any major discrepancies that are to be found.
For instance, are the DE genes identified in this study a subset of those found by Ishikawa? Related to the above point, another major component missing in this manuscript is a bird's eye view comparison to other relevant large-scale sequencing based studies.
As authors pointed out in the Discussion, there are examples of endosymbioses that are based on nitrogen and carbohydrate nutrients, including those, such as coral-Symbiodinium, Paramecium-Chlorella, and salamander-green alga, that have been investigated by transcriptome approach.
We the comments extremely helpful and constructive. Of course, innate immunity plays a major role. Therefore, in addition to the metabolic aspects of the symbiotic interactions described, we now added a chapter to present and discuss the differentially expressed immunity genes. We also followed all other suggestions in revising our work.
In order to provide a better overview of the Hydra-Chlorella symbiosis, we added new analyses of all differentially expressed Hydra genes and the gene loss in the native symbiont Chlorella A We also expanded the Discussion on generality and specificity of animal-algal symbiotic systems and its genome evolution and extended Figure 8 with a comparison between Hydra-Chlorella symbiosis and coral- Symbiodinium symbiosis. We hope our responses provided below satisfactorily address all issues you have noted.
Thank you for this excellent and constructive comment. In the previous version of our manuscript, we specifically focused on the metabolic genes, which are differentially expressed in native green hydra compared to both aposymbiotic hydra and hydra with non-native symbiotic alga NC64A.
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