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Both Ambystoma maculatum (a–d) and Ambystoma gracile (e–h) have intracapsular Oophila amblystomatis algae living in association with their embryos (c, g) despite their geographic isolation (b, f). While this alga can enter tissues and cells of A. maculatum (d white, arrowheads), it has never been observed embedded in A. gracile tissues (h, N = 10 clutches). Range maps in (b) and (f) Copyright © 2018 NatureServe (IUCN et al. 2004).
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A unique symbiosis occurs between embryos of the spotted salamander (Ambystoma maculatum) and a green alga (Oophila amblystomatis). Unlike most vertebrate host-symbiont relationships, which are ectosymbiotic, A. maculatum exhibits both an ecto- and an endo-symbiosis, where some of the green algal cells living inside egg capsules enter embryonic tis...
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... One of the known examples of photosymbiosis involves the photosynthetic microalgae that form symbioses with bacteria [78], ciliates [79], fungi [80], plants [81], and vertebrates including sloths [82] and spotted salamanders [8,83]. A unique photosymbiotic association occurs between microalgae, Oophila amblystomatis, and embryos of the spotted salamander whereby microalgae enter the cells of the salamander during early development [84]. Given the symbiotic associations of photoautotrophic microalgae with vertebrates, researchers have recently begun to explore the potential of the microalgae-based photosymbiotic oxygenation strategy for tissue engineering and regeneration [85]. ...
As the field of tissue engineering and regenerative medicine progresses, the possibility for artificial organs to restore normal tissue functions seems to become more feasible. However, a major challenge in the long-term culture of the engineered tissues is the lack of adequate oxygenation. The photosynthetic supply of oxygen (O2) for tissues and organs using photoautotrophic microorganisms has been explored recently in both in vitro and in vivo studies. The biofabrication of photosymbiotic scaffolds using biomaterials, photosynthetic microorganisms, and human cells has shown constant generation of O2 in response to light illumination while avoiding hypoxic conditions. This emerging strategy of photosymbiotic oxygenation is potentially an attractive approach to overcome the need of adequate oxygenation in tissue engineering and regenerative medicine. This Perspective aims to present an overview on the applications of photoautotrophic microorganism-enabled oxygenation strategies for overcoming hypoxia-related challenges in tissue engineering and regenerative medicine.
... Oophila algae live in multiple habitats, including pondwater (Bishop et al. 2021), inside amphibian eggs (Kim et al. 2014), and within A. maculatum tissues and cells (Kerney 2011). In order to find promoters that would drive expression across known physiological conditions, gene expression data from prior studies (Burns et al. 2017;Kerney et al. 2019) was analyzed to find transcripts that were expressed at a high level and with a low variance across multiple conditions relevant to the symbiosis including cultured algae, algae from salamander egg capsules, and intracellular algae (Fig. 1). A 2kbp region upstream of the start codon of candidate genes was recovered from the O. amblystomatis genome assembly. ...
... The algal strain that we transformed has no specific modifications with respect to wild-type O. amblystomatis. It contains a cell wall and is competent for invasion of host salamanders (Kerney et al. 2019). We show expression of transgene mRNA and protein accumulation from eight independent transfection trials in addition to showing transgenic protein function by way of antibiotic resistance and GFP fluorescence. ...
... For comparison, three genes with highly variable and condition dependent expression levels are shown (RNAPb, RPS3, RPL6). Data were collected fromBurns et al. 2017 andKerney et al. 2019 and include transcripts level of capsular algae (pink color), intracellular algae (blue color) and cultured algae (green color) ...
The ability to conduct reverse genetic studies in symbiotic systems is enabled by transgene expression and transformation of at least one partner. The symbiotic relationship between the yellow spotted salamander, Ambystoma maculatum, and the green alga, Oophila amblystomatis, is a unique model of vertebrate-algae symbiosis. Despite over 130 years of scientific study, there are still many open questions in this symbiosis. Transgene expression in one partner will accelerate research into the symbiotic relationship. In this paper we describe a tool and method for expression of foreign DNA in, and presumed transformation of, the alga O. amblystomatis. We successfully introduced heritable antibiotic resistance to algal cultures, and observed expression of a green fluorescent reporter protein in all transfected and presumably transformed algal populations. The outcomes of this work enable genetic manipulation of the symbiotic alga O. amblystomatis, allowing direct testing of hypotheses derived from gene expression or genomic studies that will usher in a deeper understanding of the A. maculatum-O. amblystomatis symbiotic system.
... Kim et al. (2014) showed that algae harvested from capsules of A. maculatum and those of its congener A. gracile are in the Oophila clade, but they largely segregate phylogenetically by host. Other than A. maculatum, among known amphibian egg mass hosts of Oophila, only A. gracile has been investigated carefully for evidence of intra-cellular invasion; there was none (CDB unpublished observations; Kerney et al. (2019). Using reciprocal coculture experiments with algae derived from A. maculatum or A. gracile embryos Kerney et al. (2019) tested the fidelity of the invasive phenotype. ...
... Other than A. maculatum, among known amphibian egg mass hosts of Oophila, only A. gracile has been investigated carefully for evidence of intra-cellular invasion; there was none (CDB unpublished observations; Kerney et al. (2019). Using reciprocal coculture experiments with algae derived from A. maculatum or A. gracile embryos Kerney et al. (2019) tested the fidelity of the invasive phenotype. They found that, whereas algae from A. gracile can (at a substantially lower frequency) also invade A. maculatum embryos, algae from neither A. gracile nor A. maculatum can invade A. gracile embryos. ...
The unicellular green alga Oophila amblystomatis forms a symbiosis with embryos of the spotted salamander Ambystoma maculatum. The discovery of intracellular invasion of some host cells by the symbiont has raised questions about benefits for the symbiont or the host, including the possibility of vertical symbiont transmission. To determine the provenance of algal cells that reproduce inside individual egg capsules I collected female salamanders from two different ponds in 2019 and 2021 and induced them to oviposit in custom-manufactured bins containing either autoclaved or non-autoclaved pond water. In both years, algae accumulated in all egg masses oviposited in non-autoclaved water. In 2019 no algae accumulated in egg masses oviposited in autoclaved water and in 2021 a few egg capsules in two egg masses from two different bins accumulated algae near the end of the experiment. For the 2021 experiment, I used PCR with Oophila-specific primers and PacBio sequencing of the 18S rRNA gene to confirm that green egg capsules contained Oophila sp; there was no evidence of algal DNA in capsules from egg masses having no visible algae. These results support previous evidence that algae are derived from pond water, but the appearance of algae in 2021 in egg masses laid in autoclaved water suggests that additional sources of algae are possible.
... Salamander genome information remains incomplete, in part because of its size ( 31 Gb) (Burns et al. 2017, Licht andLowcock 1991), which renders whole-genome sequencing difficult. The molecular classification of the symbiont algae is also obscure (Kerney et al. 2019). Thus, as a robust tool for uncovering the molecular mechanism, transgenic experiments are not presently feasible. ...
... gracile), Jefferson (A. jeffersonianum), tiger (A. tigrinum), and Japanese black (Hynobius nigrescens) salamanders (Kerney 2011, Muto et al. 2017, and wood (Lithobates sylvaticus) and red-legged (Rana aurora) frogs (Gilbert 1942, Kerney 2011, Muto et al. 2017, Melo Clavijo et al. 2018, Kerney et al. 2019. In each case, algae have been observed within egg capsules, but no further in-depth research has yet been conducted to determine if they truly have a symbiotic relationship. ...
Photosymbiosis occurs in symbiotic relationships between heterotrophs and photosymbiotic organisms, where a photosynthetic symbiont provides photosynthate to a host. While almost all known host animals are invertebrates, experiments have determined that artificial symbiotic relationships can be established between vertebrates and phototrophs. The ability to generate photosymbiotic relationships in vertebrates has important applications for medical treatments and commercial resource production.
... Large egg masses, in particular, pose challenges to developing embryos with respect to oxygen availability within eggs [74]. The embryos of wood frogs (Rana sylvatica) [75], northern red-legged frogs (Rana aurora) [75], European agile and common frogs (Rana dalmantina and R. temporaria, respectively) [76], spotted and northwestern salamanders (Ambystoma maculatum and A. gracile, respectively) [8,77,[87][88][89][90], potentially Ambystoma jeffersonianum [78], and the Japanese black salamander, Hynobius nigrescens [79], all form associations with a clade of green alga, Oophila amblystomatis, within their egg capsules. Of these, the most well studied association is that between A. maculatum and O. amblystomatis. ...
... Recent research on this association has focused on: (1) the algal symbiont, including its molecular taxonomy [75,76,81,82], origin [83,84], material benefits [85,86], and latent effects on embryo physiology [87]; and (2) the host embryo, including host specificity [88], morphology impacts [12,86], and spatio-temporal factors of the habitat [82,84]. The discovery of an intracellular component to this association [8] brought a new frontier in this symbiosis and raised new questions (e.g. ...
Photosymbioses, intimate interactions between photosynthetic algal symbionts and heterotrophic hosts, are well known in invertebrate and protist systems. Vertebrate animals are an exception where photosynthetic microorganisms are not often considered part of the normal vertebrate microbiome, with a few exceptions in amphibian eggs. Here, we review the breadth of vertebrate diversity and explore where algae have taken hold in vertebrate fur, on vertebrate surfaces, in vertebrate tissues, and within vertebrate cells. We find that algae have myriad partnerships with vertebrate animals, from fishes to mammals, and that those symbioses range from apparent mutualisms to commensalisms to parasitisms. The exception in vertebrates, compared with other groups of eukaryotes, is that intracellular mutualisms and commensalisms with algae or other microbes are notably rare. We currently have no clear cell-in-cell (endosymbiotic) examples of a trophic mutualism in any vertebrate, while there is a broad diversity of such interactions in invertebrate animals and protists. This functional divergence in vertebrate symbioses may be related to vertebrate physiology or a byproduct of our adaptive immune system. Overall, we see that diverse algae are part of the vertebrate microbiome, broadly, with numerous symbiotic interactions occurring across all vertebrate and many algal clades. These interactions are being studied for their ecological, organismal, and cellular implications. This synthesis of vertebrate–algal associations may prove useful for the development of novel therapeutics: pairing algae with medical devices, tissue cultures, and artificial ecto- and endosymbioses.
... Additionally, it has been argued that the host embryos may provide nitrogenous compounds to algae (Goff and Stein 1978), yet this hypothesis remains controversial (Bianchini et al. 2012;Small et al. 2014). The close symbiotic association of Oophila with its amphibian hosts even leads to algal cells invading host embryonic tissues and cells in the spotted salamander (Ambystoma maculatum), and thus constitutes a unique example of endosymbiosis in vertebrates (Kerney et al. 2011(Kerney et al. , 2019. ...
... This clade had a somewhat isolated (phylogenetic) position within Chlamydomonadales, and besides, the clutch-associated algae contained only three isolates of free-living algae. Isolates of this clade have been consistently considered as O. amblystomatis, which is regarded as the numerically most abundant alga in A. maculatum (Jurga et al. 2020) and A. gracile (Kerney et al. 2019;Marco and Blaustein 2000) egg capsule chambers. These O. amblystomatis isolates have been used in studies of gene expression (Burns et al. 2017;Kerney et al. 2019), carbon fixation (Burns et al. 2020), and host-symbiont fidelity (Kerney et al. 2019). ...
... Isolates of this clade have been consistently considered as O. amblystomatis, which is regarded as the numerically most abundant alga in A. maculatum (Jurga et al. 2020) and A. gracile (Kerney et al. 2019;Marco and Blaustein 2000) egg capsule chambers. These O. amblystomatis isolates have been used in studies of gene expression (Burns et al. 2017;Kerney et al. 2019), carbon fixation (Burns et al. 2020), and host-symbiont fidelity (Kerney et al. 2019). As an exception, a few isolates from A. maculatum egg masses, along with environmental samples, were assigned to Chlamydomonas gloeophila; however, C. gloeophila was suggested to represent low abundance green algae occurring in these egg masses that outcompeted Oophila under agar media growth conditions in culture (Kim et al. 2014). ...
Amphibian clutches are colonized by diverse but poorly studied communities of micro-organisms. One of the most noted ones is the unicellular green alga, Oophila amblystomatis , but the occurrence and role of other micro-organisms in the capsular chamber surrounding amphibian clutches have remained largely unstudied. Here, we undertook a multi-marker DNA metabarcoding study to characterize the community of algae and other micro-eukaryotes associated with agile frog ( Rana dalmatina ) clutches. Samplings were performed at three small ponds in Germany, from four substrates: water, sediment, tree leaves from the bottom of the pond, and R. dalmatina clutches. Sampling substrate strongly determined the community compositions of algae and other micro-eukaryotes. Therefore, as expected, the frog clutch-associated communities formed clearly distinct clusters. Clutch-associated communities in our study were structured by a plethora of not only green algae, but also diatoms and other ochrophytes. The most abundant operational taxonomic units (OTUs) in clutch samples were taxa from Chlamydomonas , Oophila , but also from Nitzschia and other ochrophytes. Sequences of Oophila “Clade B” were found exclusively in clutches. Based on additional phylogenetic analyses of 18S rDNA and of a matrix of 18 nuclear genes derived from transcriptomes, we confirmed in our samples the existence of two distinct clades of green algae assigned to Oophila in past studies. We hypothesize that “Clade B” algae correspond to the true Oophila , whereas “Clade A” algae are a series of Chlorococcum species that, along with other green algae, ochrophytes and protists, colonize amphibian clutches opportunistically and are often cultured from clutch samples due to their robust growth performance. The clutch-associated communities were subject to filtering by sampling location, suggesting that the taxa colonizing amphibian clutches can drastically differ depending on environmental conditions.
... It has been suggested that the entry of the algae into the salamander cells resembles endosymbiotic relationships in the phylum protozoa (Kerney et al. 2011;Lane and Archibald 2008). Additionally, the interaction has a high fidelity, i.e., only the designated species can form both appropriate ectosymbiotic interaction and endosymbiotic dispersion within the salamander developmental tissues (Kerney et al. 2019). Hypothetically, it could be a result in EGT. ...
Endosymbiosis or symbiogenesis is a process where a cell hosts another cell that is acquired through phagocytosis or natural entry of the cell within its cytoplasm. Endosymbiosis has a profound effect on the survival of the host cell by conferring nutritional and/or biosynthetic advantage. Therefore, attempts of artificial endosymbiosis have become one of the most challenging projects in synthetic biology. In this paper, we review the process of endosymbiosis, its levels, requirements and mechanisms. We then review the unique cases of ‘natural endosymbiosis’. Furthermore, we describe and evaluate the recent cases of attempted artificial endosymbiosis. Subsequently, we assess the potential barriers to the possibility of endosymbiosis of highly evolved cell types such as mammalian cells that are known for their high inflexibility towards hosting potentially even the most ‘benign endosymbionts’. The paper concludes with possibilities and methodologies that may have not been evaluated or tried in the past, but may be used to increase the chance of artificial endosymbiosis of host cells such as those from mammalian origin that are not permissive to even benign endosymbionts. Artificial endosymbiosis is worth revisiting in this post-genomic, synthetic biology era because the tools and techniques currently available at our disposal have significantly advanced to make this grand challenge a possibility.
... There is some disagreement as to which lineage or lineages of algae found in amphibian egg capsules correspond to the originally described binomial O. amblystomatis (Kim et al., 2014;Nema et al., 2019;Kerney et al., 2019;Jurga et al., 2020). Symbiotic association with algae has been documented in A. maculatum (Spotted Salamanders), Ambystoma gracile (Northwestern Salamanders), Lithobates sylvatica (Wood Frogs), Lithobates aurora (Northern Red-legged Frogs), and more recently Hynobius nigrescens (Japanese Black Salamanders), with ribosomal sequences from each clustering together phylogenetically (Kim et al., 2014;Muto et al., 2017). ...
... Symbiotic association with algae has been documented in A. maculatum (Spotted Salamanders), Ambystoma gracile (Northwestern Salamanders), Lithobates sylvatica (Wood Frogs), Lithobates aurora (Northern Red-legged Frogs), and more recently Hynobius nigrescens (Japanese Black Salamanders), with ribosomal sequences from each clustering together phylogenetically (Kim et al., 2014;Muto et al., 2017). Uncertainty exists as to the phylogenetic relationships between algae within this clade, the number of subclades, if the clade is monophyletic or even made up of a single species, and if there is a reciprocal relationship between algal lineage and host species (Kim et al., 2014;Kerney et al., 2019;Nema et al., 2019;Jurga et al., 2020). Herein, our study will deal with any alga that symbiose with amphibian eggs that may fall within the Oophila clade (Kim et al., 2014;Jurga et al., 2020). ...
... Vertical transmission involves the symbionts being passed from one generation to the next via parental reproductive system (usually maternal), and horizontal transmission requires the entrance of the symbiont from the external environment in each generation (Bright and Bulgheresi, 2010). Evidence and arguments supporting both mechanisms have been provided throughout the literature (Gilbert, 1942;Gatz, 1973;Kerney, 2011;Kerney et al., 2011Kerney et al., , 2019. ...
The symbiosis between developing Spotted Salamanders (Ambystoma maculatum) and the unicellular alga known as Oophila amblystomatis appears to be mutualistic, with involved parties trading apparent benefits within the salamander egg capsule, but the spatiotemporal ecology of the interaction has yet to be thoroughly explored. Using newly developed primers for quantitative polymerase chain reaction (PCR), we sampled 149 salamander egg masses of various ages from six breeding ponds in northeast Alabama. We documented the presence of algal DNA within egg capsules throughout the developmental process. Regression via linear mixed model estimation showed a positive relationship between the age of egg masses and the probability of algal DNA detection and a negative trend when comparing egg-mass age and qPCR cycle quantification value. These trends indicate an increase in both the probability that a given egg will contain algal DNA and the amount of algal DNA an egg contains, if present, as age increases. We found no effects of pond, site within pond, or year of sampling in either case.
... Particular attention has been given to the conspicuous association between the common spotted salamander of North America (Ambystoma maculatum) and its O. amblystomatis symbiont (Orr, 1888;Gilbert, 1942). While recent studies have focused on the unique facultative endosymbiotic association of algal cells inside the embryo host (Kerney et al., , 2019Burns et al., 2017), there is a long history of research into the ecto-symbiotic association between freeliving Oophila inside the embryonic egg capsule (Kerney, 2011). This intracapsular Oophila has a role in oxygenating the egg capsule microenvironment (Gilbert, 1942(Gilbert, , 1944Bachmann et al., 1986;Pinder and Friet, 1994;Mills and Barnhart, 1999;Bianchini et al., 2012) and potentially in removal of nitrogenous waste from the host (Goff and Stein, 1978;Bianchini et al., 2012;Small et al., 2014). ...
... The result suggests that some algae persist through the washes but living A. maculatum embryos do not actively accrue additional measurable photosynthate from lab-cultured algae in these conditions. We note that although cultured algae are in a different transcriptional state from intracapsular algae (Kerney et al., 2019), lab-cultured algae are able to interact with and invade A. maculatum embryos ex situ, similar to algae in the intracapsular environment (Kerney et al., 2019). In this study, decapsulated embryos incubated in the dark accumulated significantly more fixed carbon ( Figure 2B) compared to embryos incubated with pre-labeled algae (p < 0.001) or embryos killed with glutaraldehyde prior to incubation with bicarbonate (p < 0.001). ...
... The result suggests that some algae persist through the washes but living A. maculatum embryos do not actively accrue additional measurable photosynthate from lab-cultured algae in these conditions. We note that although cultured algae are in a different transcriptional state from intracapsular algae (Kerney et al., 2019), lab-cultured algae are able to interact with and invade A. maculatum embryos ex situ, similar to algae in the intracapsular environment (Kerney et al., 2019). In this study, decapsulated embryos incubated in the dark accumulated significantly more fixed carbon ( Figure 2B) compared to embryos incubated with pre-labeled algae (p < 0.001) or embryos killed with glutaraldehyde prior to incubation with bicarbonate (p < 0.001). ...
The unique symbiosis between a vertebrate salamander, Ambystoma maculatum, and unicellular green alga, Oophila amblystomatis, involves multiple modes of interaction. These include an ectosymbiotic interaction where the alga colonizes the egg capsule, and an intracellular interaction where the alga enters tissues and cells of the salamander. One common interaction in mutualist photosymbioses is the transfer of photosynthate from the algal symbiont to the host animal. In the A. maculatum–O. amblystomatis interaction, there is conflicting evidence regarding whether the algae in the egg capsule transfer chemical energy captured during photosynthesis to the developing salamander embryo. In experiments where we took care to separate the carbon fixation contributions of the salamander embryo and algal symbionts, we show that inorganic carbon fixed by A. maculatum embryos reaches 2% of the inorganic carbon fixed by O. amblystomatis algae within an egg capsule after 2 h in the light. After 2 h in the dark, inorganic carbon fixed by A. maculatum embryos is 800% of the carbon fixed by O. amblystomatis algae within an egg capsule. Using photosynthesis inhibitors, we show that A. maculatum embryos and O. amblystomatis algae compete for available inorganic carbon within the egg capsule environment. Our results confirm earlier studies suggesting a role of heterotrophic carbon fixation during vertebrate embryonic development. Our results also show that the considerable capacity of developing A. maculatum embryos for inorganic carbon fixation precludes our ability to distinguish any minor role of photosynthetically transferred carbon from algal symbionts to host salamanders using bicarbonate introduced to the egg system as a marker.
... There is some disagreement as to which lineage or lineages of algae found in amphibian egg capsules correspond to the originally described binomial O. amblystomatis (Kim et al., 2014;Nema et al., 2019;Kerney et al., 2019;Jurga et al., 2020). Symbiotic association with algae has been documented in A. maculatum (Spotted Salamanders), Ambystoma gracile (Northwestern Salamanders), Lithobates sylvatica (Wood Frogs), Lithobates aurora (Northern Red-legged Frogs), and more recently Hynobius nigrescens (Japanese Black Salamanders), with ribosomal sequences from each clustering together phylogenetically (Kim et al., 2014;Muto et al., 2017). ...
... Symbiotic association with algae has been documented in A. maculatum (Spotted Salamanders), Ambystoma gracile (Northwestern Salamanders), Lithobates sylvatica (Wood Frogs), Lithobates aurora (Northern Red-legged Frogs), and more recently Hynobius nigrescens (Japanese Black Salamanders), with ribosomal sequences from each clustering together phylogenetically (Kim et al., 2014;Muto et al., 2017). Uncertainty exists as to the phylogenetic relationships between algae within this clade, the number of subclades, if the clade is monophyletic or even made up of a single species, and if there is a reciprocal relationship between algal lineage and host species (Kim et al., 2014;Kerney et al., 2019;Nema et al., 2019;Jurga et al., 2020). Herein, our study will deal with any alga that symbiose with amphibian eggs that may fall within the Oophila clade (Kim et al., 2014;Jurga et al., 2020). ...
... Vertical transmission involves the symbionts being passed from one generation to the next via parental reproductive system (usually maternal), and horizontal transmission requires the entrance of the symbiont from the external environment in each generation (Bright and Bulgheresi, 2010). Evidence and arguments supporting both mechanisms have been provided throughout the literature (Gilbert, 1942;Gatz, 1973;Kerney, 2011;Kerney et al., 2011Kerney et al., , 2019. ...
Using 18S rDNA we created a developmental timeline showing when algae invade the eggs of Spotted Salamanders
