Symbiotic interactions in the holobiont Hydra
Jay Bathia and Thomas С. C. Bosch
Introduction
Life has evolved in a microbial world. The event of successful symbiosis was a pivotal step that resulted in the origin of a eukaryotic cell. Organisms, therefore, are considered metaorganisms comprised of the macroscopic host and synergistic interdependence with bacteria, archaea, fungi and numerous other microbial and eukaryotic species including algal symbionts (Bosch and McFall-Ngai 20П). The interactions between all the partners are affected by the prevailing environmental conditions. Understanding the mechanisms and the causality of the interactions in such a metaorganism requires model organisms where the contribution of each partner can be disentangled and functionally analyzed.
Aptly named. Hydra is known for its remarkable capacity of regeneration and non-senescence. For decades, Hydra has been used as a model organism to study the cellular pathways governing body patterning and ageing by evolutionary conserved mechanisms (Galliot 2012). However, like all other metaorganisms. Hydra hosts numerous bacterial, viral and algal symbionts (Habetha et al. 2003; Fraune and Bosch
2007; Grasis et al. 2014). For various reasons. Hydra turns out to be an excellent model system to study the interactions between these partners (Bosch 2013). Hydra being diploblastic, possess a simple body plan composed to two stem cell epithelial layers - ectoderm and endoderm, separated by a thin layer of connective tissue, mesoglea. The third stem cell lineage, the interstitial cells, is localized in the ectoderm and gives rise to the nervous system, nematocytes, gland cells and germ line cells. Towards the outside on the ectoderm, a multilayered glycocalyx harbours the bacterial symbionts.
The extensive genomic and transcriptomic data of various Hydra species can be utilized to map known pathways and look for evolutionary conserved genes. Moreover, it is also possible to genetically manipulate the host in loss/gain of function studies (Wittlieb et al. 2006; Klimovich et al. 2019). The transparent tissue facilitates visualization of cellular components without rigorous fixing and bleaching methods. Large populations of Hydra can be easily cultivated and maintained under lab conditions. Clonal propagation ensures the genetic homogeneity of the entire population. In addition, since Hydra belongs to the phylum Cnidaria which is the sister group of Bilaterians, it is an informative model to study evolutionary conserved mechanisms in metazoans. Being able to separate all the partners of the metaorganism makes Hydra suitable to study the underlying principles of the complex multiplayer interactions (Li et al. 2015, 2017; Wein et al. 2018). In this chapter, we summarize the state of the art understanding of the Hydra metaorganism, the interactions between the host and algal symbiont in Hydra viridissima, and also the interactions of the Hydra host with the bacterial symbionts.
Interactions between Hydra viridissima and the Chlorella photobiont
Location and transmission of the photobiont
Symbiotic Chlorella algae are present intracellularly in the endodermal epithelial cells. This location points to a high level of co-dependence and a rather complex cellular machinery involved in the maintenance of the symbiosis. Inside the Hydra endothelial cells, the algae are held towards the base of the cells (Jolley and Smith 1978) (Figure 5.1A). This compartmentalizes an endodermal cell into two zones: digestive zone, which is the apical part of the cell responsible for phagocytosis and digestion of the food vesicles and the symbiotic zone, which is at the base of the cell where the symbiotic algae are present (McNeil 1981; McNeil et al. 1981). Inside the host cell, each algal cell is surrounded by a host-derived vesicle membrane that acts as an interface for the biotrophic exchange of nutrients. This ‘peri-symbiotic’ membrane is the result of the phagocytosis by which the symbiont is taken up (McNeil 1981; Davy et al. 2012).
The peri-symbiotic membrane is congruent to the membrane of a phagosome. The resulting structure is often referred to as a ‘symbiosome’ and the space between the peri-symbiotic membrane and the algae is called ‘peri-symbiotic space’ (Davy et al. 2012). Being present intracellularly demands a need for successful transfer of the symbiont to the next generation. Chlorella algae can be transmitted horizontally as well as vertically. The host has a capacity to expel excess of living and preferentially dividing algae actively (Baghdasarian and Muscatine 2000). Such algae can be
FIGURE 5.1 Hydra viridissima/Chlorella symbiosis. (A) Each endothelial epithelial cell harbours about 30 Chlorella algae (red auto fluorescence) in the basal part below the host nucleus (stained green). (Image courtesy: Jay Bathia and Alexander Klimovich. Zoological Institute. Kiel University.) (B) Summary of symbiotic interactions between Hydra and Chlorella A99. During light conditions, Chlorella A99 performs photosynthesis and produces maltose (Mai), which is secreted into the Hydra symbiosome where it is possibly digested to glucose (Glue), shown in red. The sugar induces expression of Hydra genes encoding glutamine synthetase (GS), Na/Pi transporter (NaPi) and Spotl4. GS catalyses the condensation of glutamate (Glu) and ammonium (NH4+) to form glutamine (Gin), which is used by Chlorella as a nitrogen source. Since the sugar also upregulates 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 also to the ectoderm (red broken line) and there induces the expression of GS and Spotl4. (Modified from Hamada. M.. et al. 2018. ELife 7. doi: 10.7554/ eLife.35122.) later be taken up from the environment by an aposymbiotic host to reestablish symbiosis. Vertical transmission takes place during the asexual reproduction phase because the endodermal cell layer of the mother polyp is in continuation with that of the developing bud. which ensures the transfer of algae in the resulting bud (Jolley and Smith 1980). Interestingly, symbiotic algae can also be transmitted vertically to the offspring through germ-line maternal cells (Campbell 1990).
Mutual benefits
The mutual exchange of metabolites between the host and the photobiont results in a phenotypic benefit for both the partners. The population growth rate of symbiotic animals is faster than the aposymbiotic animals (Habetha et al. 2003; Ishikawa et al. 2016; Hamada et al. 2018). Moreover, the symbiotic animals can survive for a longer period under starvation as compared to the aposymbiotic animals (Ishikawa et al.
2016). It has also been observed that the presence of algae increases the frequency of host oogenesis (Habetha et al. 2003). Since oogenesis is typically an energetically expensive process, the transport of sugar from the algae may aid in overcoming the cost of oogenesis. Interestingly, any attempt to culture the endosymbiotic algae in vitro consistently failed. This underlines the strong dependence of the algae on the host. Apart from being nutritionally beneficial, the algae can protect the host under environmental stress. A recent study (Ye et al. 2019) indicates that if symbiotic algae were acclimated to higher temperatures, they provide survival benefits to the Hydro holobiont at higher temperatures. There are a number of studies that utilized the ability of Hydra viridissima to establish symbiosis with foreign endosymbiotic algae such as Chlorella NC64A (Dorling et al. 1997; Kodama and Fujishima 2015; Hamada et al. 2018). Chlorella NC64A is a native endosymbiont of the protist Paramedian bursaria. Chlorella NC64A can release sugar to the host only in low amounts, resulting in lower population growth rate, almost equivalent to aposymbiotic animals (Hamada et al. 2018), compared to polyps harbouring the native photobiont Chlorella A99. This further indicates the importance of harbouring a native symbiont.
Establishment and maintenance of the Chlorella-Hydra symbiosis
To establish a new symbiosis, the aposymbiotic host engulfs the algae through phagocytosis. This results in the formation of a phagosome enclosing the algae. It is acidified by the fusion with acidosomes (Allen and Fok 1983). Only when the engulfed algae is a true symbiont is the fusion of lysosome inhibited, as indicated by the absence of acid phosphatase activity in the late phagosome stage (Hohman et al. 1982). The recognition of symbiotic algae occurs probably by the presence of microbe- associated molecular patterns (MAMPs). Recent comparative transcriptomic studies indicate the putative involvement of the Toll/Interleukin-1 receptor (TIR). a known pattern recognition receptor (PRR) and C-type lectin, to be involved in recognizing the MAMPs of a symbiotic algae (Hamada et al. 2018).
The physiological conditions, and in particular the pH, experienced by an endosymbiotic alga are very different from those experienced by a free-living alga. The pH of the peri-symbiotic space is maintained between 4 and 5. This is crucial because only at this low pH is the algae shown to be able to release maltose (Cernichiari et al. 1969; Dorling et al. 1997). The maltose releasing capacity is unique to endosymbiotic algae. In plants and free-living algae, during nighttime, the stored starch in chloroplasts is mobilized to the cytoplasm in the form of maltose, but never secreted to the extracellular space (Lu and Sharkey 2006). This may point to a maltose transport machinery on the membrane of the symbiotic algae similar to the membrane of the chloroplast. Another important characteristic of a successful mutualistic relationship is to control the proliferation of the endosymbiont. There is evidence that Hydra control the number of algae per cell by regulating the pH of the peri-symbiotic space (Muscatine and Neckelmann 1981; McAuley 1986; Dunn 1987). Moreover, the host is capable to actively expel any excess number of algae (Baghdasarian and Muscatine 2000).
Molecular mechanisms involved in maintaining the symbiosis
A elaborated comparative transcriptomics and genomic study of Hydra viridissima revealed the key components and pathways involved in maintaining the host-algal symbiosis (Hamada et al. 2018). To identify the host genes involved in maintaining symbiosis with the native algae, this unbiased high-throughput study (Hamada et al. 2018) included a comparison of the symbiotic host associated with the native symbiotic Chlorella A99, the aposymbiotic host and the host bearing foreign symbiotic algae Chlorella NC64A.
Comparing the native symbiotic animals with aposymbiotic animals revealed the upregulation of a number of conserved cellular pathways in symbiotic animals, which maintain the symbiosis. The transcriptomics data revealed the up-regulation of a host V-type ATPase subunit and carbonic anhydrase, involved in maintaining the low pH of the perisymbiotic space and facilitating CO, transport across the symbiosome respectively. Moreover, there is an upregulation of GLUT8-like transporters, putatively involved in transportation of the photosynthate across the peri-symbiotic membrane. The study also uncovered the up-regulation of cell- adhesion genes encoding rhamnospondin and fibrillin. These molecules might be involved in retaining the algae to the base of the endothelial cell. Moreover, a high level of peroxidase, methionine-r-sulfoxide reductase/selenoprotein and glutaredoxin might help to combat the surge in oxidative stress generated by the constant oxygen influx from the algae to the host.
A comparison of Hydra polyps, harbouring Chlorella A99, with Hydra polyps colonized by the non-native Chlorella NC64A algae showed that the interactions between the host and the algae are mainly metabolic in nature (summarized in Figure 5.1 B). The native photobiont provides the photosynthetically fixed carbon to the host in the form of maltose. This maltose is likely to be digested to its monomer glucose, which is transported to the host by transporter proteins in the symbiosome membrane. This excess sugar shifts the host metabolism to ammonium assimilation. In response to this, there is an up-regulation of host glutamine synthetase (GS-I) which is involved in the production of glutamine from ammonium and glutamate. GS-I is specific to the symbiotic state, as other isoforms are unaffected by the presence or absence of the algae. As another important stabilizer of the Hydra-Chlorella symbiosis, the sodium- dependent phosphate transport protein (NaPi) can increase the available phosphate for the host and the algae. Apart from these, a Spot_14 like domain-containing protein with a potential role in lipid metabolism, and two taxonomically restricted genes (TRGs) are also up-regulated. Exposing polyps to the photosynthesis inhibitor DCMU or culturing them at constant darkness, results in a drastically reduced expression level of these genes, indicating that the expression of these symbiosis-specific genes is dependent on the photosynthetic activity of the symbiont.
Genome analysis of the symbiotic Chlorella A99 algae and the comparison to the genome of the free-living counterparts revealed interesting features, which helped to explain the strong dependence of the symbiont on the host (Hamada et al. 2018). Most strikingly, the symbiotic algae have lost their key nitrite/nitrate and ammonium assimilation genes. Symbiotic algae, therefore, depend on the host for fixed nitrogen in the form of glutamine. Further genomic analyses of the algae also indicated an increase in the number of amino acid transporters, supporting amino acid uptake. Finally, although all attempts to culture these algae in vitro failed, the growth of the algae can be temporarily maintained by adding CAS amino acids or glutamine as a source of nitrogen to the culture medium. This reflects the strong metabolic co-dependence of both partners and a unique adaptation of the symbiotic algae (Hamada et al. 2018).
