RICE UNIVERSITY Stress tolerance of the endosymbiotic cnidarian Hydra viridissima Contributions of the host and the symbiont

By

Siao Ye

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

Doctor of Philosophy

APPROVED, THESIS COMMITTEE

Evan Siemann Chair Amy Dunham

Evan Siemann Amy E Dunham

Sc P. Ega Meenakshi Bhattacharjee Apr Scott P Egan Apr 1 2020 Meenaskshi Bhattacharjee Scott Egan

Meenakshi Bhattacharjee Scott P Egan

Rudy Guerra Apr 13, 2020 Rudy Guerra Rudy Guerra

HOUSTON, TEXAS April 00 iii

ABSTRACT

Stress tolerance of the endosymbiotic cnidarian Hydra viridissima: Contributions of the host and the symbiont

by Siao Ye

Endosymbiosis is a universal relationship in nature, in which the symbiont is tightly integrated into the host and alters the host’s ecological niche and evolutionary trajectory. However, endosymbiosis can be easily disrupted by stress, which raises concerns about holobiont (symbiont plus host) persistence with global climate change, because massive endosymbiosis breakdown could have severe ecological consequences. The hologenome theory proposes that the symbiont and the host jointly determine the holobiont phenotype, and so rapid holobiont phenotype alteration can be achieved through changes in symbiont composition via strain acquisition, adaptation or acclimation. Previous studies have confirmed that switching symbionts can change holobionts’ stress tolerance, yet the relative roles of the symbiont and the host in controlling the holobiont phenotype are unclear as is whether symbiont adaptation or acclimation alone could allow holobionts to persist in stressful environments. My thesis addressed factors affecting stress tolerance of the holobiont green hydra (Hydra viridissima) from both the symbiont (green algae) and the host (hydra) perspectives, and experimentally explored how manipulations of the symbiont or the host alone could alter holobiont stress tolerance. Furthermore, I used agent-based modeling to simulate the holobiont adaptation via symbiont adaptation. My experimental results showed unequal contributions of the host and the symbiont to holobiont stress tolerance, with the host playing a dominant role. Furthermore, I detected positive transgenerational effects in the host. Although host traits are usually overlooked in holobiont studies, my studies suggest they are equally or even more important in determining holobiont stress iii tolerance. On the other hand, symbiont adaptation or acclimation provides the holobiont an extra route to acquire stress tolerance if the host fails to acquire it directly, and such acquired stress tolerance can be inherited. In addition, my experiments proved the feasibility of altering holobiont phenotypes by symbiont manipulations in vivo. Simulations further demonstrated that holobionts with either vertical or horizontal symbiont transmission are able to evolve solely through adaption of the symbiont. These findings support the hologenome theory and have implications for forestry, agriculture and conservation by providing insights into the determination and management of holobiont stress tolerance.

Acknowledgments

Foremost I would like to thank my advisor Evan Siemann. Thanks for his time and knowledge, and especially the flexibility and independency under his supervision, which made this thesis possible. Being his only student all these years, I received his full support on my research. I would also like to thank my committee members, Dr. Amy Dunham, Dr. Meenakshi Bhattacharjee, Dr. Scott Egan and Dr.

Rudy Guerra, who all supported my Ph.D study and provided important suggestions and feedback on my research in time.

My special thanks give to Zhu Liu, my undergrad classmate and a close friend who is studying at University of South California, USA. He contributed significantly to my last chapter and helped me with the coding.

For the supply of the hydra and algae in my lab, I would like to thank Dr.

Daniel Martinez (Pomona College, Claremont, CA, USA), Dr. Hiroshi Shimizu

(National Institute of Genetics, Mishima, Japan) and Dr. David Dunigan (University of Nebraska Lincoln, NE, USA). Also thanks Dr. Daniel Wagner and Dr. Jacob

Robinson for their allowance of using their equipment.

Next, I would like to thank Dr. Adrienne Correa, Dr. Tom Miller and Dr.

Volker Rudolf, who all gave constructive suggestions during our discussions. I also want to thank people from my department who helped with my research: Brad

Ochocki instructed me in R, Amanda shore and Therese Lamperty proofread my manuscript, Krishna Badhiwala and Eric Wice demonstrated how to monitor

v animals, Linyi Zhang took care of my hydra when I was away, and Shannon Carter discussed about the agent-based modeling. A special thanks to Patrick Clay who took me and other students to watch “Guardian of the Galaxy” the first day we arrived, and organized activities all these years, which made my life not only fill with exams and lab work. I owe further thanks to my lab members, Stephen Truch and

Maria Meza-Lopez, who supported my first year at Rice. I am also grateful for my undergrad research assistants, who all contributed their time and effort to the lab work.

Finally, I cannot be more thankful to my family, my wife Yuan Song, my parents, Yubin Ye and Lezhen Zhao, and my sister Simiao Ye, as well as my dog Tutu.

Your supports produce another “Dr.” in this world.

Contents

Acknowledgments ...... iv Contents ...... vi List of Figures ...... ix List of Tables ...... xi Introduction ...... 1 1.1. Holobionts under stress ...... 1 1.2. Impacts of the symbiont on the holobiont stress tolerance ...... 3 1.2.1. Net impacts of symbionts ...... 3 1.2.2. Existing symbiont composition alteration ...... 4 1.2.3. Symbiont adaptation and acclimation ...... 5 1.3. Impacts of the host on the holobiont stress tolerance ...... 9 1.4. Heritability of symbiont-mediated traits ...... 20 1.5. Aim of the study ...... 20 Thermal tolerance in green hydra: identifying the roles of algal endosymbionts and hosts in a freshwater holobiont under stress ...... 23 2.1. Introduction ...... 24 2.2. Methods ...... 27 2.2.1. Study Species and Culture Conditions ...... 27 2.2.2. Strains with Natural Symbionts ...... 28 2.2.3. Strains without Symbionts ...... 29 2.2.4. Different Algal Strains Associated with a Single Hydra Strain ...... 29 2.2.5. Control Hydra Strains Re-Associated with Their Original Symbionts ...... 30 2.2.6. Heat Shock Treatment ...... 30 2.2.7. Data Analysis ...... 31 2.3. Results ...... 31 2.3.1.1. Establishment of Aposymbiotic Hydra, Novel Hydra, Control Hydra .. 31 2.3.2. Strain Specific Thermal Tolerance in Four Types of Hydra ...... 32 2.3.3. Thermal Tolerance in Symbiotic and Aposymbiotic Hydra ...... 35 2.3.4. Algae Thermal Tolerance ...... 35

vii

2.4. Discussion ...... 36 2.4.1. Relative Contribution of the Host and the Endosymbiont to Hydra Thermal Tolerance ...... 36 2.4.2. Adaptation Capability of Endosymbiotic Species from the Hologenome Perspective ...... 38 2.4.3. Partner Selection in Endosymbiosis Establishment ...... 41 2.5. Conclusions ...... 42 Symbiont mutagenesis and characterization as a potential method to mitigate climate change impacts on holobionts: a study on green hydra Hydra viridissima ...... 43 3.1. Introduction ...... 44 3.2. Materials and Methods ...... 48 3.2.1. Study species and culture conditions ...... 48 3.2.2. Algae mutation and selection ...... 50 3.2.3. Algae UV-B resistance tests ...... 51 3.2.4. Regreened hydra UV-B tolerance test ...... 52 3.2.5. Statistical analyses ...... 52 3.3. Results ...... 54 3.4. Discussion ...... 58 3.5. Conclusions ...... 63 Thermal plasticity of a freshwater cnidarian holobiont: detection of trans- generational effects in asexually reproducing hosts and symbionts ...... 65 4.1. Introduction ...... 66 4.2. Materials and Methods ...... 70 4.2.1. Focal organisms ...... 70 4.2.2. Population Growth Rate Measurement ...... 71 4.2.3. Contraction Activity Monitoring ...... 72 4.2.4. Thermal Tolerance ...... 73 4.2.5. Statistical Analysis ...... 74 4.3. Results ...... 76 4.4. Discussion ...... 81 4.5. Conclusions ...... 87 viii

An agent-based modeling of adaptation in holobionts with different transmission modes ...... 89 5.1. Introduction ...... 90 5.2. Method ...... 97 5.2.1. Purpose ...... 97 5.2.2. Scales and variables ...... 98 5.2.3. Scheduling ...... 98 5.2.4. Design Concepts ...... 102 5.2.5. Simulation scenarios ...... 103 5.2.6. Statitical analysis ...... 104 5.3. Results ...... 104 5.3.1. Test case ...... 104 5.3.2. Increasing temperature ...... 105 5.4. Discussion ...... 110 References ...... 119 Appendix A ...... 144

List of Figures

Figure 2.1: Hydra survival curves of (A) original, (B) aposymbiotic, (C), novel, and (D) control groups following one-hour heat shock at 35°C...... 32

Figure 2.2: Relationships between green hydra thermal tolerance and host/endosymbiont effects...... 34

Figure 2.3: NC64A algae chlorophyll Optical Density (OD) values following one- hour heat shock at 35°C...... 35

Figure 3.1: Experiment flowchart of (A) algae mutagenesis and selection, (B) hydra- algae reassociation...... 49

Figure 3.2: The change in OD at 663 nm (ln [ODt=48hrs / ODt=0hrs]) for different non- selected (dark symbols or lines) or selected (light symbols or lines) populations of algae from different cultures in (A) control and (B) UV-B test conditions. Means ± se. Means with the same letters did not differ in post-hoc tests. * indicates p<0.05. (C)

Algal tolerance to UV-B in test conditions (ln ([ODUVB,t=48hrs / ODUVB,t=0hrs] /

([ODcon,t=48hrs / ODcon,t=0hrs])) estimated by randomization...... 55

Figure 3.3: The survival of hydra associated with different non-selected (dark symbols or lines) or selected (light symbols or lines) populations of algae...... 56

Figure 3.4: The correlation (line) between algal change in OD in UV-B (Fig 2B) and Cox survival coefficient for different non-selected (dark circles) or selected (light squares) populations of algae...... 57

Figure 4.1: Growth of symbiotic hydra (green symbols and lines) and aposymbiotic hydra (black symbols and lines) at 18°C before acclimation (controls - squares), acclimating at 30°C (triangles), or acclimated at 30°C for two months (circles)...... 76

Figure 4.2: Variation in contraction frequency of symbiotic and aposymbiotic hydra in non-acclimated state (time 0hr, 18°C), acclimating state (time 1 to 12hrs, transferred to 30°C), and acclimated states (time 1440hrs, 30°C) with time...... 78

Figure 4.3: Thermal tolerance of hydra heat shocked at 38°C in states of 1) non- acclimated controls at 18°C (triangles). 2) acclimating at 30°C (circles). 3) acclimated after two months at 30°C (circles). 4) deacclimating that were transferred back to 18°C (squares). 5) deacclimated after two months back to 18°C (squares)...... 79

x

Figure 4.4: Survival of symbiotic and aposymbiotic starving hydra at 30°C after acclimation...... 80

Figure 4.5: Thermal tolerance of reassociated algae and hydra: (1) Both hydra and algae experienced acclimation (HH.HA). (2) Both hydra and algae were not exposed to high temperature (LH.LA). (3) Only hydra experienced acclimation (HH.LA). (4) Only algae experienced acclimation (LH.HA)...... 81

Figure 5.1: Fitness curve of symbiont impacts on the holobiont ...... 75

Figure 5.2: Fixation of holobionts under selection neutral condition. Blue lines are Vs and red lines are Hs...... 80

Figure 5.3: Gene values of H and V in (a) random inheritance model at step=0.001, mutation_sd=0.1, (b) optimized inheritance model at step=0.001, mutation_sd=0.01...... 81

Figure 5.4: Fitness/Extinction time of Vs and Hs in (a) random inheritance model, (b) optimized inheritance model. Large step (high temperature increasing speed) and small mutation_sd (low mutation variance) are likely to lower holobiont fitness and lead to extinction...... 107

Figure 5.5: V’s average proportion during the simulation of (a) random inheritance, (b) optimized inheritance...... 108

Figure 5.6: Symbiont fitness score variance (a) within the host, (b) between the host...... 109

List of Tables

Table 4.1 Growth rates of symbiotic and aposymbiotic hydra with different acclimation and symbiosis statuses (ln[#] = a + k×days)...... 77

Chapter 1

Introduction

This chapter has been re-formatted, modified, and reprinted from Ye S,

Siemann E. 2020. Endosymbiont-mediated adaptive responses to stress in holobionts (in “Symbiosis: Cellular, Molecular, Medical and Evolutionary Aspects” ed. Kloc M, Springer Nature Series in “Results and Problems in Cell Differentiation”).

1.1. Background

Endosymbiosis is a ubiquitous phenomenon found in nature, which refers to two distantly related species that exhibit close physical contact in the form of one species living inside the other [1]. While a comprehensive definition of endosymbiosis involves interactions that range from parasitic to mutualistic, the latter receives increasing attention in recent years because beneficial symbionts have been found to have significant ecological and evolutionary impacts on their hosts [2]. Due to the strong interdependency between the host and the symbiont,

2 researchers have proposed that the two parties can be viewed as one entity called the holobiont and that they jointly determine the holobiont phenotypes [3].

Because the holobiont’s existence relies on well-being of both the host and the symbiont, stress response of one party could negatively affect the other. Thus species that are highly endosymbiosis-dependent could be especially vulnerable to climate change or other types of environmental change because they may experience mutual downfall [4]. Such vulnerability of holobionts to stress has been observed in various endosymbiotic species. For example, corals (marine invertebrates in the class Anthozoa) that host endosymbiotic algae (Symbiodinium dinoflagellates) have experienced massive bleaching (loss of algae) in past decades, because abnormal environmental conditions such as high temperature or high salinity damages the algae [5, 6]. Similarly, short-term exposure can deplete insects of their endosymbiotic bacteria (that provide essential amino acids to sap-feeding insect hosts [7]), and reduce their fecundity [8, 9]. Whether holobionts can rapidly shift their stress tolerance is receiving increasing attention, and the answers may shed light on holobiont adaptation and future holobiont management.

According to the hologenome theory, holobiont stress tolerance could be rapidly altered through adaptive responses of the symbiont [3]. Compared to the host, the endosymbiont is more likely to shift holobiont stress tolerance in the short term because of their larger populations and shorter generation times. Thus more mutations may occur in symbionts than in hosts for a given period, and adaptive evolution is more likely to take place in symbionts and to occur more quickly [10, 3

11]. In addition, the holobiont may be able to respond to stress without any change in the host, but rather through changes in their endosymbiont composition (e.g., species or strains), which could be the key to holobiont persistence in changing climates if host adaptation is slow. From a holobiont perspective, as long as the endosymbiont affects the holobiont phenotype, any genetic or non-genetic changes in the endosymbiont could influence holobiont fitness, and have ecological or evolutionary consequences. In this review, we discuss symbionts’ impacts on holobionts’ stress tolerances, and demonstrate symbiont-mediated adaptive responses in holobionts.

1.2. Endosymbionts’ contributions to holobiont stress

While the nutrient supplement function of the endosymbiont has long been well-recognized, the topic of endosymbiont-mediated stress tolerance is gaining more attention since researchers discovered that endosymbionts can play a significant role in host development or physiology [12, 13]. The holobiont could exhibit distinctive stress tolerance from the host or the endosymbiont alone, providing a distinct, emergent holobiont phenotype that is subjected to selection.

For example, lichens exhibit significant higher stress tolerance compared to that of the alga and the fungus forming the holobiont [14], which could probably be explained by the complex morphology formed by the two partners, and their complicated biochemical interactions [15]. Association with endosymbionts seems to alter the ecological niches of hosts, which further facilitates their diversification into diverse environments [16]. 4

However, having endosymbionts does not always enhance holobiont stress tolerance. Whether the presence of endosymbionts has a positive or negative impact on the holobiont’s stress tolerance could depend on the species as well as the stress type. In other words, even the same type of endosymbiont could have opposite impacts on the holobiont fitness with regard to different kinds of stress. Here we would like to introduce three of the most well-studied endosymbiotic systems to show how endosymbionts could affect holobiont stress tolerance.

1.2.1. Plant-fungi system

Almost all plants in nature are associated with fungi that can be categorized into mycorrhizal fungi or endophytic fungi [17]. Mycorrhizal fungi usually reside in plant roots and rhizosphere, while the endophytic fungi may be found throughout the entire plant [18]. Although mycorrhizal fungi are well-known for their nutrient supplementary function, both mycorrhizal and endophytic fungi confer abiotic stress tolerance to plants, which enhances their performance under drought, heat, or high salinity [19–22]. Surprisingly, such positive impacts could be transgenerational even when the offspring are fungus-free, apparently through epigenetic modifications of the host [23, 24]. In some plants, endosymbionts are necessary for the plant to grow in their native habitats. For example, Dichanthelium lanuginosum grasses growing in geothermal regions are colonized by fungal endophyte Curvularia protuberata, without which they will not be able to survive the high temperature [20]. Similarly, the coastal dunegrass Leymus mollis hosts the fungal endophyte Fusarium culmorum, which confers salt tolerance to the host [22]. 5

Endophytes confer such tolerance by regulating plant osmotic pressure and stomata, stimulating generation of antioxidative enzymes, or supplementing anti-stress chemicals [18, 25, 26]. Mycorrhizal fungi are also able to alleviate abiotic stress such as drought or high salinity by restructuring the soil and improving water/nutrients uptake [27–29]. For example, AMF-infected Cucumis sativus cucumber plants contain higher concentrations of inorganic nutrients and up-regulated activity of antioxidant enzymes than AMF-free controls under salt stress, which contributes to their higher biomass in such environments [30]. Compared to endophytic or mycorrhizal fungus-free plants, plants hosting these types of fungi usually exhibit improved tolerance of stressful environmental conditions such as salinity, heat, drought, or heavy metals. [25, 29].

1.2.2. Aquatic organism-algae system

Invertebrate animals or protists in oligotrophic aquatic ecosystems often form symbioses with algae that provide fixed forms of carbon in exchange for physical protection and nutrients [31, 32]. These organisms have wide distributions from freshwater to marine ecosystems, and some of the most well studied organisms include Porifera (sponges), Cnidaria (corals, sea anemone, hydra),

Ciliophora (paramecium and other ciliated protists) and Foraminifera (amoeboid protists) [33–35]. The interactions between algae and these organisms have received great attention in the past few decades because some of the algae-bearing animals, corals for example, are keystone species in aquatic ecosystems and are subjected to anthropogenic disturbances [5, 36, 37]. Symbiosis breakdown in these 6 organisms may cause ecological cascades, because they are important producers or degraders that support growth of other creatures, and promote biodiversity [38–41].

There are two main types of algae in photosynthetic symbiosis: Chlorella species (green algae) and Symbiodinium species, which are mainly found in freshwater and marine water respectively [33]. Under normal conditions, the algae fix carbon and release oxygen [31, 42]; however, the photosynthetic system could be disrupted under heat stress or strong irradiance, which produces harmful reactive oxygen species (ROS) that can lead to symbiosis breakdown [32, 43]. Studies in corals and sea anemone have revealed that stressed algae in the animal hosts accumulate ROS and induce cell apoptosis [44, 45]. The hosts then digest or expel algae as a protective mechanism, which is known as bleaching [46–49]. In contrast, host cells without algae showed limited stress response under elevated temperature

[50, 51]. Coral larvae that are free of algae lack transcriptional responses to oxidative stress during heat stress [52]. In addition, Yakovleva et al. [53] showed that algal-free coral larvae had higher survival compared to algal-bearing larvae in heat stress experiments. Green hydra (Hydra viridissima) also exhibited a similar pattern with presence of algae having non-positive impacts on hydra thermal tolerance, although contrasting results were found in Paramecium species with positive effects of algae presence [54, 55]. The high sensitivity of photosynthetic systems to heat and light stress indicates photosynthetic endosymbionts could negatively impact the host under certain abiotic stresses, and make the holobiont more susceptible to environmental perturbations [43, 56]. On the other hand, studies on Paramecium bursaria and sea anemone show that endosymbiotic algae 7 can protect the holobiont from UV radiation by both physical and biochemical mechanisms and hosts that had their endosymbionts removed received more damage [57–59] . Thus whether the presence of photosynthetic endosymbionts increases or decreases the host stress tolerance depends on the stress types.

1.2.3. Insect-bacteria system

Insects are a highly diverse and abundant group of animals and many of them associate with endosymbionts [60, 61]. Usually these endosymbionts provide nutrients such as vitamins or amino acids to hosts, but they are also involved in host development and immunity [61]. For example, almost all aphids host Buchnera aphidicola bacteria which supplement essential compounds lacking in their diet [62].

In weevils, a symbiotic bacteria Nardonella species is required for the development of hard cuticles [63]. Being small poikilotherms, insects are susceptible to temperature changes [8], and temperature fluctuations could easily affect endosymbionts that reside in them [64]. Impacts on these endosymbionts could potentially alter whole insect communities and ecosystems as effects cascade through food webs [65].

The endosymbiotic bacteria in insects are generally categorized into two groups: primary endosymbionts and secondary endosymbionts [66]. Primary endosymbionts are obligate microbes that are transmitted vertically, from mothers to offspring; secondary endosymbionts are facultative microbes that are not necessary for insect survival, and can be acquired horizontally [67, 68]. Primary endosymbionts usually have coevolved with hosts for millions of years, and are 8 integrated into their hosts’ life cycles [68]. The long-term coevolution with hosts drives the endosymbiont to lose genes that are redundant with those of the host, so the bacteria might lack necessary genes to maintain functional stability on their own

[16]. The specialized organs (bacteriocytes) hosting these endosymbionts are also susceptible to stress [69]. Thus these key endosymbionts could be easily lost during environmental stress, and negatively impact insect hosts [70]. For example, high temperature has shown similar effects as antibiotics on an obligate bacterial symbiont in the stinkbug Nezara viridula, suppression of which reduces the host growth and body size [64]. Aphids that were exposed to 25-30°C also experienced declines in their obligate endosymbiotic Buchnera bacteria [71]. Moreover, such declines in endosymbionts could have further negative impacts on offspring, because the transmission rate is also largely reduced [67]. Given that global climate change could dramatically reshape current climate, such as increasing the occurrence of extreme temperatures, insects that are highly dependent on primary endosymbionts would face great challenges and may even become extinct [70]. In contrast, species that are not associated with primary endosymbionts may be more likely to expand their ranges and potentially become invasive [70]. While the primary endosymbionts may fail during environmental stress, secondary endosymbionts may rescue the hosts from the loss of their obligate endosymbionts

[8, 72]. For instance, aphids that were eliminated of their primary Buchnera endosymbionts due to heat stress experienced decreases in their fecundity, but inoculation of secondary endosymbionts partially restored their fertility [71]. The secondary endosymbionts could also confer novel traits to hosts and improve their 9 stress tolerance [73, 74]. Because the primary endosymbionts and secondary endosymbionts differ in their evolutionary history and intimacy with hosts, their impacts on host stress tolerance can be quite distinctive [8, 66, 68].

1.3. Adaptive responses of holobionts to stress via

endosymbionts

Because of the intimacy between the endosymbiont and the host, the holobiont phenotype can be largely influenced by the former in addition to the host

[3]. Presence of endosymbionts could confer novel traits to hosts and enable the holobionts to expand into new niches, but could also increase their sensitivity to stress [1, 70, 75]. Because large scale endosymbiosis breakdowns have already occurred with severe ecological consequences, one key question is whether holobionts are able to alter their stress tolerance via symbiont changes to alleviate the stress [47, 76–78]. Here we discuss about some common strategies of holobionts responding to stress.

1.3.1. Impacts of existing symbiont composition alteration

Hosts could associate with multiple potential endosymbiont genotypes or species, and one-to-one obligate endosymbiosis is rare in nature [79, 80]. The diverse symbionts serve as a pool from which holobionts could choose optimal partners, because these symbionts exhibit distinctive phenotypes in vitro and thus the tolerance they can confer to hosts [81, 82]. According to the hologenome theory, holobionts can achieve rapid shifts in stress tolerance by taking advantage of 10 existing symbiont variation in three ways: 1) symbiont amplification (change in relative abundances of diverse associated symbionts); 2) novel symbiont acquisition

(uptake of novel symbionts horizontally, i.e. from the environment or other holobionts); 3) horizontal gene transfer (transfer of genes among symbionts or between symbionts and hosts) [3].

1.3.1.1. Endosymbiont amplification

Holobionts hosting diverse endosymbionts can adjust to environmental changes through amplification of those that maximize holobiont fitness [3].

Endosymbiont abundances within hosts could be comparable to that of host cells, and their diversity could be higher than expected because low-density microbes are hard to detect [83]. For example, critical endosymbiotic bacteria that are part of diverse assemblages may be rare in cicadas and at risk of failing to be passed on to offspring by their mothers, so these types of bacteria may be passed on in relatively much higher numbers than bacteria groups that have low diversity (and high per strain abundance) in the host [84]. In corals, Symbiodinium diversity is high not only in the community but can also be high within an individual [85–87]. Because coral thermal tolerance is largely determined by the endosymbionts, amplification of certain types of endosymbionts may greatly alter the holobiont stress tolerance

[83, 88, 89]. Researchers have revealed that Symbiodinium species vary in their stress tolerances [90]. Heat sensitive Symbiodinium produce more ROS when subjected to heat stress, and their growth rates in vitro are also suppressed [82, 90,

91]. Corals associated with these sensitive Symbiodinium are more likely to bleach 11 under stress, but they can become more resistant by shifting to stress tolerant endosymbionts [92, 93]. Jones et al. [92] found the relative abundance of stress tolerant endosymbionts increased at both community and individual levels after stress occurred even though they were previously dominated by stress sensitive endosymbionts. In addition, corals that become dominated by stress tolerant endosymbionts after stress can better withstand future stress [94]. In fact, the improved stress tolerance is not only a result of increase in tolerant endosymbionts, but also due to elimination of intolerant endosymbionts [48, 95]. Similarly, insects usually harbor heat sensitive endosymbionts under normal conditions, which are crucial to maintain physiological function [68, 70]. While these primary endosymbionts are eliminated by heat stress, some tolerant secondary endosymbionts could survive and rescue the host from malfunction [8, 69, 71].

Amplification of rarer but more stress tolerant endosymbionts appears to be a common method for holobionts to achieve higher stress tolerance and persist in climate change but the dominance of stress sensitive endosymbionts in benign conditions suggests a trade-off between stress tolerance and other potentially beneficial traits [81].

1.3.1.2. Novel endosymbiont acquisition

Novel endosymbiont acquisition is another mechanism for holobionts to alter their stress tolerance [3, 96]. Many endosymbionts have a free-living stage in their life cycles, during which they are released into the environment and can be picked up by hosts [97, 98]. For example, endosymbiotic fungi are rich in soil, and settled 12 plants can form associations with nearby fungi [17]. Corals are able to conduct both vertical transmission and horizontal transmission, through the latter they are able to pick up novel endosymbionts [99, 100]. Such acquisition of novel endosymbionts appears to be common and may be key to species radiation [96, 101]. Chong and

Moran [102] discovered Geopemphigus aphids lost their obligate Buchenera endosymbionts over evolutionary time, but established endosymbiosis with a new type of bacteria. Because endosymbionts vary in their response to stress, tolerances conferred to hosts by different endosymbionts could also vary. Marine ecologists have proposed that such endosymbiont switching could rescue corals from extinction under climate change, because some Symbiodinium species are more resistant to stress [93, 103]. Plants are also able to acquire stress tolerance by associating with specific endosymbiotic fungi [17]. Curvularia isolates (a type of fungal endophyte) from geothermal regions can confer thermal tolerance to fungal- free plants by establishing endosymbiosis with hosts, which also suggests stress tolerance could be acquired horizontally [20]. In aphids, artificial inoculation of two facultative endosymbionts increased their resistance to heat stress, in contrast, those that only hosted obligate endosymbionts had significantly lower reproduction success [71]. These evidences support that association with novel endosymbionts may alleviate stress in holobionts.

1.3.1.3. Horizontal gene transfer among endosymbionts

Horizontal gene transfer is an important force that shapes the endosymbiont phenotype, and thus the holobiont phenotype [78, 104]. Such gene exchanges are 13 common in both eukaryotes and prokaryotes, which indicates all kinds of endosymbionts, from bacteria to algae and fungi, are able to acquire novel traits horizontally [105, 106]. Research in plant-associated endosymbionts revealed signs of intra-clade horizontal gene transfer, which affected their secondary metabolism

[107]. In an endosymbiotic Chlorella species from Paramecium, researchers detected gene framents that might have originated from a virus [108]. Because viruses can insert into multiple kinds of species and package additional cellular genes, the endosymbiont could acquire novel genes indirectly [109, 110]. For example, three phages were found to contain functional photosynthesis genes probably from different cyanobacteria, and cyanobacteria form endosymbioses with a variety of species, including corals, algae, and fungi [15, 111–113]. In addition, horizontal gene transfer could happen between endosymbionts and their hosts.

Moran and Jarvik [114] discovered aphids may acquire its carotenoid biosynthetic genes from fungi, and Chapman et al. [115] detected that the hydra genome contains several bacteria-originated genes. Horizontal gene transfer could contribute to endosymbiont stress tolerance and impact holobiont persistence under stress [116].

1.3.2. Impacts of symbiont adaptation and acclimation

In addition to utilizing existing genetic variation in endosymbionts, holobionts could respond to environmental stress through adaptation and acclimation responses in their microbial partners [116]. Adaptation and acclimation are two major mechanisms that alleviate organisms’ stress in changing environments, which could take place in both the endosymbiont and the host [3, 14

117–119]. However, microbes have higher plasticity and adaptability compared to host animals [120], which suggests holobiont stress tolerance can be altered simply through symbiont acclimation or adaptation, which is especially critical for species hosting low-diversities of endosymbionts [97, 121].

1.3.2.1. Adaptation

Adaptation involves genetic changes due to natural selection on the population level, which results in a shift in the mean phenotypes. Because microbes typically have very short generation times, their adaptation could be relatively fast compared to hosts [3, 122]. For instance, the doubling time of Symbiodium species is typically between days to months, but up to years for their coral hosts [23, 93,

122]. In addition, the endosymbiont population size far exceeds that of the host because one individual holobiont could contain thousands of endosymbionts [123–

125]. That is to say, mutations are more likely to occur in the endosymbionts and provide opportunity for adaptation.

Studies on endosymbiotic species have confirmed intra-species adaptation in endosymbionts. Rodriguez et al. [22] found the same fungal species may or may not confer stress tolerance based on habitats they were collected from. Fungi that were isolated from geothermal habitats or costal habitats conferred thermal tolerance or drought/salinity tolerance to host plants respectively. However, the same species collected from non-stressful habitats did not improve the plant stress tolerance when they were introduced into hosts. Local adaptation of endosymbionts was also observed in corals. Howells et al. [126] compared the stress response of a general 15

Symbiodinium type from a warmer reef and a control reef. They found even after multiple asexual generation of cultivation in the lab, endosymbionts from the warmer region exhibited lower stress response to elevated temperature than endosymbionts from the control, such as higher survival and quantum yield. The endosymbiotic bacteria Buchnera in aphids is also found to control aphid thermal tolerance [9]. A single nucleoid mutation in Buchnera determines the thermal tolerance of aphids, and both high-temperature adapted and low-temperature adapted strains can be found in the wild population. This suggests aphids hosting different Buchnera strains might diverge in their ecological niches and adapt to different environments. Attempts have also been made to test for adaptability of endosymbionts. Chakravarti et al. [127] conducted experimental evolution on

Symbiodinium species, and they found the endosymbionts were able to improve their stress tolerance over 2 years. They raised algae in vitro at an elevated temperature of 31°C for about 80 generations, and observed improvement in growth rate and decrease in ROS production when subjected to stress. However, they did not detect significant differences in thermal tolerance between corals inoculated with the selected algae and corals with wild type algae. Because the host may fail to adapt fast enough to keep up with environmental change, endosymbiont adaptation may allow the holobiont more time to adapt.

1.3.2.2. Acclimation

Acclimation is a reversible physiological process of organisms to deal with stress, which involves no genetic changes but adjustments in expression [128]. The 16 word “acclimation” is sometimes used interchangeably with the word

“acclimatization”, but the latter usually refers to a more comprehensive concept, and in holobiont studies it may include a broader range of microbial responses, such as endosymbiont amplification or switching [116, 128]. Here we use the term

“acclimation” to describe such phenotypic plasticity without genetic changes.

Acclimation has been observed in holobionts by investigating their responses under stress over prolonged periods. For example, corals exposed to high temperature are able to adjust their metabolisms in both the host and the endosymbiont, which reflects non-genetic adaptive responses to stress in both parties [129, 130]. In another acclimation experiment, while coral calcification rate was strongly suppressed under short-term high CO2 exposure, it was able to recover over a long-term exposure [131]. In addition, the holobiont can acclimate not only to increases in stress intensity but also in stress variability [132], and the acclimatory ability differs among populations [133]. In fact, acclimation can effectively assist holobionts to persist in stress because preconditioning to sublethal stress improves the holobiont’s tolerance towards subsequent acute stress [118,

134].

More precise detection of acclimatory responses in the endosymbiont has been demonstrated recently. Takahashi et al. [135] inspected how long-term elevated temperature affected photosynthesis systems of Symbiodinium species in vitro under heat stress, and they found acclimation to high temperature could improve thermal stability of the endosymbiont. Transcriptome analysis of 17 endosymbionts in corals and clams reveals gene expressions related to photosynthesis, membrane lipid synthesis and ROS scavenging are mostly altered

[136–139]. In contrast, Palumbi et al. [117] analyzed the transcriptome profiles of the host and the endosymbiont in corals reciprocally transplanted between high variation pools and low variation pools. They found the acclimation was mainly attributed to the host instead of to the endosymbiont. Symbiodinium species seem to be more responsive to higher stress because their transcriptome changes increased with the concentration of a toxic pollutant [140].

However, studies on how acclimation in the endosymbiont affects holobiont stress tolerance are still limited, because most studies measured responses of the holobiont where the symbiont and the host are usually treated as an intact entity.

Due to the intimacy between the symbiont and the host, it is usually difficult to isolate the net impacts of endosymbiont acclimation. Recently, Bui and Franken discovered endosymbiotic fungi can acclimate to heavy metal stress (Zn) in culture, and confer Zn tolerance to host plants when introduced into hosts. Together these studies provide evidence that acclimation in endosymbionts alone could improve stress tolerance in holobionts, and we suggest more attention to investigating this relatively poorly studied topic.

1.4. Heritability of symbiont-mediated traits

Whether the holobiont can be considered an integrated unit subject to selection is under debate, because it addresses a core question: what is a biological 18 individual? [141–143]. A key question that should be answered is how heritable the microbes associated with hosts are, which is critical for the reproduction of an adapted holobiont. Some researchers have claimed that host associated microbes could be shaped simply by opportunity and environmental filters, and the transmission fidelity is low [141, 144]. In this review, we focus specifically on endosymbiont-host interactions, in which the endosymbiont is a necessary component of the holobiont phenotype.

There are two endosymbiont transmission modes in holobionts: vertical and horizontal transmission. In vertical transmission, offspring inherit symbionts directly from their parents [145]. An extreme case could be mitochondria, which originated from bacteria but can now be found in most eukaryotes [145–147]. Green hydra transmit their algae to offspring during both sexual and asexual reproduction, and offspring are born with algae from parents [148, 149]. Aphids are able to selectively transmit their obligate Buchnera endosymbionts to offspring through complicated cellular regulations while preventing facultative endosymbionts from transmission [150]. Vertically transmitted endosymbionts can be considered as heritable extended genomes with high fidelity, and traits conferred by this type of symbiont are also heritable [77]. In contrast, holobionts conducting horizontal transmission may not receive symbionts from their parents, but may take up random ambient symbionts [98]. The disassociation between the host and the symbiont could result in the loss of acquired beneficial symbionts, and random symbiont colonization of the host would disqualify the holobiont being a selection unit [79]. Surprisingly, recent studies reveal inheritance is not completely random 19 in horizontally transmitted endosymbionts. A study on coral found both vertically and horizontally transmitted symbionts are partially heritable, for which the host genes might be responsible [151, 152]. They discovered corals inherited shuffled

(changes in relative abundances) endosymbionts years after bleaching happened, which indicates holobionts are able to maintain the stability between endosymbionts and hosts. Schweitzer et al. [153] also discovered non-random assembly of below ground microbes associated with plants, suggesting an unknown mechanism of microbe recruitment. In a stinkbug, offspring are able to pick up the beneficial symbiont from the environment every generation, which demonstrates a high fidelity between the microbe and the host can be kept in horizontal transmission [154]. All of these suggest potential inheritance of horizontally transmitted symbionts, even if it is not as perfect as that of vertically transmitted ones.

Perhaps more important questions are: do both transmission modes allow holobionts to adapt to changing environment, and how do they impact the adaptability? Roughgarden [155] used a simulation model to show that holobionts using either transmission mode are able to evolve, regardless of whether there is disassociation between the host and the symbiont. Yet it does not tell how holobionts may react to increasing environmental stress with different transmission modes. On one hand, vertical transmission allows direct inheritance of adaptive endosymbionts in offspring, but it imposes a strong bottleneck effect on the population of endosymbionts the offspring could get [97, 125]. Endosymbiont diversity could be depleted over time, and the microbes may become maladaptive 20 once the ambient condition changes [156]. For example, psyllid insects that conduct vertical transmission have low diversity in their bacterial endosymbionts, and in some cases become clonal within the host [121]. Loh et al. [157] also showed higher endosymbiont genetic heterogeneity among populations exists in vertically transmitted corals than in horizontally transmitted corals. On the other hand, horizontal transmission enables the holobiont to associate with a great variety of endosymbionts, so they may have access to more adaptive endosymbionts [98, 100].

But these holobionts might lose adaptive endosymbionts between generations by chance, which reduces their fitness over time [79]. Research in human culture transmission could be enlightening, in which similar concepts such as vertical transmission and oblique transmission of cultures or knowledge are studied [158].

Some of the major findings are that vertical culture transmission is favored under stable conditions, while horizontal transmission is favored under fluctuating conditions [159–162]. Nevertheless, there could be fundamental differences between culture transmission and endosymbiont transmission, and research in a holobiont context is needed.

1.5. Aim of the study

The aim of this thesis was to explore whether green hydra Hydra viridissima is an appropriate system to study holobiont stress tolerance, and can be used to identify the relative roles of the symbiont and the host. In addition, the thesis also addressed factors that could alter holobiont stress tolerance, and provides 21 simulation result to verify the feasibility of holobiont adaptation via the symbiont adaptation.

Chapter 2 investigated the net impacts of symbiotic algae on green hydra thermal tolerance, and researched if strain-specific thermal tolerance existed in the host and the symbiont separately. By comparing the relative thermal tolerance of six original hydra-algae strains with derived aposymbiotic hydra and derived hydra that only different in their symbionts, I found thermal tolerance variation existed in both the host and the symbiont, with the host playing a dominant role in holobiont thermal tolerance, while the algal symbiont had a non-positive impact. This study confirmed green hydra is an appropriate system to further study holobiont stress tolerance because the symbiont and the host can be manipulated independently.

Chapter 3 explored whether symbiont treatment in vivo can alter its stress tolerance and therefore stress tolerance of the holobiont after the symbiont is inoculated into the host. I mutated algae with UV-C and selected them with UV-B to see if algal UV-B tolerance could be altered via mutagenesis and artificial selection. I then tested UV-B tolerance of hydra associated with these treated algae and control algae. The results showed that mutagenesis rather than artificial selection altered the stress tolerance of the symbiont and the holobiont, and algal UV-B tolerance was positively correlated to that of the hydra hosting correspondent algae. I also discussed the potential application of this method.

Chapter 4 studied whether acclimation could take place either in the symbiont or the host, and checked how the presence of symbiont would affect the 22 holobiont acclimation and deacclimation. I showed that hydra with acclimated algae and/or host exhibited significant increase in their thermal tolerance. Having symbionts although may reduce initial thermal tolerance and cost the holobiont more time to become acclimated, it does not affect the acclimation rate or maximum thermal limits after acclimation. In fact, presence of the symbiont is quite beneficial when the heat stress co-occurs with starvation.

Chapter 5 employed agent-based modeling to investigate whether vertically transmitted and horizontally transmitted holobionts can adapt to increasing temperature, and compared the relative fitness of these two types of holobiont in various conditions. It turns out that both holobiont types are able to improve their stress tolerance solely relying on symbiont adaptation. However, the relative fitness of these two holobiont types depends on whether the host is able to recognize tolerant symbionts or not. Vertically transmitted holobionts adapt faster when symbiont inheritance is completely random, but are inferior to horizontally transmitted holobionts when the host can pick out optimized symbionts.

23

Chapter 2

Thermal tolerance in green hydra: identifying the roles of algal endosymbionts and hosts in a freshwater holobiont under stress

This chapter has been re-formatted, modified, and reprinted from: Ye S,

Bhattacharjee M, Siemann E. 2019. Thermal tolerance in green hydra: Identifying the roles of algal endosymbionts and hosts in a freshwater holobiont under stress.

Microbial Ecology 77: 537–545.

Abstract

It has been proposed that holobionts (host-symbiont units) could swap endosymbionts, rapidly alter the hologenome (host plus symbiont genome) and increase their stress tolerance. However, experimental tests of individual and combined contributions of hosts and endosymbionts to holobiont stress tolerance are needed to test this hypothesis. Here, we used six green hydra (Hydra viridissima) strains to tease apart host (hydra) and symbiont (algae) contributions to thermal tolerance. Heat shock experiments with: 1) Hydra with their original symbionts, 2) 24

Aposymbiotic hydra (algae removed), 3) Novel associations (a single hydra strain hosting different algae individually), 4) Control hydra (aposymbiotic hydra re- associated with their original algae), showed high variation in thermal tolerance in each group. Relative tolerances of strains were the same within original, aposymbiotic and control treatments, but reversed in the novel associations group.

Aposymbiotic hydra had similar or higher thermal tolerance than hydra with algal symbionts. Selection on the holobiont appears to be stronger than on either partner alone, suggesting endosymbiosis could become an evolutionary trap under climate change. Our results suggest that green hydra thermal tolerance is strongly determined by the host, with a smaller, non-positive role for the algal symbiont.

Once temperatures exceed host tolerance limits, swapping symbionts is unlikely to allow these holobionts to persist. Rather, increases in host tolerance through in situ adaptation or migration of pre-adapted host strains appear more likely to increase local thermal tolerance. Overall, our results indicate green hydra is a valuable system for studying aquatic endosymbiosis under changing environmental conditions, and demonstrate how the host and the endosymbiont contribute to holobiont stress tolerance.

2.1. Introduction

The interaction between symbionts and hosts is receiving increasing attention as microorganisms have been found to be critical in host life cycles.

Symbionts are in such close relationships with hosts that they are potentially able to manipulate host physiology and modify host development [12, 13]. Thus the 25 holobiont (host-symbiont unit) phenotype is not only affected by the host but also affected by the symbiont. Indeed, there has been tremendous progress in recognizing the significant role of endosymbionts in determining holobiont traits

[126, 163–165].

One of the important traits affected by endosymbionts is stress tolerance, which shapes the holobiont niche [66, 71, 126]. For example, endophyte-infected plants exhibit changes in morphology and secondary metabolites compared with endophyte-free plants, and have higher resistance to both biotic and abiotic stress

[17, 22, 166]. With enhanced stress tolerance, these plants have broader environmental tolerances and geographic ranges [167, 168]. However, the close relationship between endosymbiont and host can link negative effects accrued by stress from one partner to the other, potentially increasing vulnerability to global climate change [169]. Thus, it is critical to understand how stress tolerance is determined in holobionts.

Rosenberg and Zilber-Rosenberg [170] suggested changes in either symbiont or host genes can alter holobiont genetic composition (hologenome) and in turn the holobiont phenotype. One of the most well studied groups regarding the endosymbiont’s role in holobiont stress tolerance is coral. In past decades, corals have experienced massive bleaching when they expel their endosymbiotic algae due to warming sea temperatures but some corals are less likely to bleach than others [5,

171, 172]. Buddemeier et al. [48, 173] hypothesized that different algae types confer different levels of thermal tolerance to hosts, and bleaching provides an opportunity 26 for corals to adapt to rising temperature by establishing associations with novel, more heat tolerant algae. The high diversity of endosymbionts are thus considered sources for holobiont adaptation, and symbiont swapping may enable holobiont persistence under stress [103]. Variation in algae stress tolerance has been shown and it has been confirmed that algae can associate with novel hosts [95, 174]. Algal strains that are more prevalent in harsh environments tend to exhibit higher thermal tolerance, and corals that switch to these algae can become more resistant to stress [93]. This indicates endosymbionts can affect holobiont stress tolerance, and changing symbionts can alter holobiont tolerance. Yet the host role in holobiont tolerance is typically overlooked, even though indirect evidence suggests intrinsic differences in hosts could cause holobiont stress tolerance variation [37, 175]. To date, however, it remains unclear whether the symbiont or the host plays a more significant role in determining holobiont tolerance.

Aquatic photosynthetic endosymbiosis may be a valuable model for studying this question because of its significance in ecosystems as well as the ease of manipulation [34, 40, 176]. Compared with endosymbionts in plants or arthropods, algae in aquatic invertebrates can be directly observed and isolated. In addition, some host species are able to survive without the symbionts, such as Paramecium bursaria [58]. This allows testing how presence of symbionts affects stress tolerance in the lab, which has often found symbionts improved host survival under stress [54,

57, 58, 177]. However, less has been done to investigate symbiont-specific and host- specific tolerances, as well as their impacts on holobiont stress tolerance respectively. 27

Here, we performed a set of experiments with green hydra (Hydra viridissima) to disentangle the contributions of symbiont (algae) and host (hydra) to holobiont thermal tolerance. In particular, we expected symbionts and hosts would vary in stress tolerance and holobiont stress tolerance would be determined in an additive way. We used heat as the stress factor because destabilization of endosymbiosis is common during temperature fluctuation [8]. The goal was not only to identify the relative roles of the symbiont and host in determining the holobiont stress tolerance phenotype but also to evaluate whether changing symbionts is a way for this, and perhaps other, aquatic endosymbiotic species to persist in novel environmental conditions.

2.2. Methods

2.2.1. Study Species and Culture Conditions

Green hydra Hydra viridissima (Anthomedusae: Hydridae) is a freshwater invertebrate that forms stable endosymbiosis with green algae (Chlorella sp.). The symbiotic algae reside in the gastrodermal epithelial cells of the hydra, and provide carbohydrates to the host. In return, algae obtain nitrogen from the host [178, 179].

Green hydra can survive in an aposymbiotic state (free of endosymbionts) as a heterotroph if enough food is available and can re-establish endosymbiosis with algae after bleaching [180]. By manipulating both the hydra and the algae, including creating novel associations, we can study endosymbiosis both from the host’s perspective and from the endosymbiont’s perspective. 28

2.2.2. Strains with Natural Symbionts

We used six strains of H. viridissima collected across a wide geographic range in the experiments. The green hydras were collected in local ponds or streams from following locations: 1) Two strains, 1695C and GBR01b, were acquired from Dr.

Daniel Martinez at Pomona College, CA. They were collected in Chile and Great

Britain, respectively. 2) Four strains were acquired from different commercial suppliers who collected them in California (Niles Biological Inc., Sacramento, CA),

Massachusetts (Connecticut Valley Biological Supply Company, Southampton, MA), or Nevada (Carolina Biological Supply Company, Burlington, NC), and a long-term cultured strain of unknown geographic origin (VWR International, Radnor, PA). We kept all hydra in petri dishes in a constant 18°C lab. We cultured them in hydra medium [181], fed them brine shrimp (Brine Shrimp Direct, Inc., Ogden, Utah) three times a week, and changed the medium weekly. We used a 25-Watt 55.88 cm long fluorescent light set 40 cm above the dishes for illumination, providing a 12:12 hour light:dark cycle [181]. The animals were acclimated to these conditions for 3 weeks before the experiment started.

The endosymbiotic algae Chlorella variabilis (NC64A) was kindly provided by

Dr. David Dunigan. This algal strain was isolated from Paramecium bursaria and was cultivated in Modified Bold's Basal Medium [182]. 29

2.2.3. Strains without Symbionts

We derived aposymbiotic hydra from the six green hydra strains by treating them with hydra solution with 1×10-8 M or 2×10-8 M 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), which is a photosynthetic inhibitor [183]. We hung a 30-watt light bulb 5 cm above the hydra for continuous illumination [181]. We fed hydra every two days and changed the medium 6 hours after each feeding. We kept the bleached hydra in cultivation for several generations and removed any re-greened hydra.

2.2.4. Different Algal Strains Associated with a Single Hydra Strain

We created new algae-hydra combinations by injecting algae from each of the six hydra strains into one aposymbiotic hydra strain (Chile) produced by Dr. Daniel

Martinez. For each source strain, we put green hydra in a tube, centrifuged them, removed the supernatant to concentrate them, and then sonicated tubes to disrupt the hydra (but left algal cells intact). We microinjected this solution into aposymbiotic hydra (Chile strain). We performed multiple injections to individual hydra over several weeks to insure inoculation success. We considered symbiont re- establishment to be successful when hydra became green again. We created an additional combination by injecting the NC64A algal strain into aposymbiotic hydra

(Chile strain). We used offspring of these re-associated hydra for the experiment. 30

2.2.5. Control Hydra Strains Re-Associated with Their Original Symbionts

We re-greened control groups hydra by associating aposymbiotic hydra with their original algae as a negative control for the DCMU treatment.

2.2.6. Heat Shock Treatment

We used a heat shock protocol to measure abiotic stress tolerance. For each strain of the four types of hydra, we transferred 25 individuals from petri dishes at

18°C to 25ml beakers with hydra solution pre-heated to 35°C in a water bath. After an hour, we took the beakers out of the bath and cooled them gradually to 18°C by sitting them on the bench top in the 18°C lab. We recorded their survival every hour for the first 8 hours after the end of the heat shock, at 12 hours, at 24 hours and thereafter every 24 hours until 120 hours (5 days). Hydra were considered dead when their bodies disintegrated.

We subjected the three replicates of 20ml NC64A algae to heat shock under same protocol but in test tubes. We extracted chlorophyll with methanol and measured optical density (OD) at 663nm wavelength to infer algae vitality. We took readings before the heat shock, then 1 day, 2 days, 4 days and 6 days after heat shock.

Our heat shock treatment was similar to those used in studies of Paramecium caudatum [184, 185] and Hydra attenuata [186]. Natural systems, especially small volume ponds, can experience similarly large changes in temperature within a few hours [187, 188]. 31

2.2.7. Data Analysis

We used interval censored survival analysis for all four types of hydra to test strain effects in R (Version 1.0.143). We performed weighted logrank tests and calculated Sun’s scores so the thermal tolerance of hydra strains could be ordered.

We compared symbiotic hydra with their original endosymbionts to aposymbiotic hydra derived from them and the control group by strains in a same way to test endosymbiosis effects and bleaching effects.

2.3. Results

2.3.1.1. Establishment of Aposymbiotic Hydra, Novel Hydra, Control Hydra

The original green hydra strains were completely bleached after one-month of DCMU treatment. We halved the DCMU concentration (to 1×10-8 M DCMU) used for the Nile strain, Connecticut strain and Carolina strain because higher concentrations (2×10-8 M DCMU) killed them.

Bleached hydra took approximately two weeks to show green patches on the body after algae were injected, and two to three more weeks for them to become completely green. In general, two or three hydra out of five to eight injected individuals turned green after injections. Carolina strain algae were the last to form symbioses with the new host strain 1695C, taking two additional weeks. 32

2.3.2. Strain Specific Thermal Tolerance in Four Types of Hydra

Figure 2.1: Hydra survival curves of (A) original, (B) aposymbiotic, (C), novel, and (D) control groups following one-hour heat shock at 35°C. Curves are plotted based on logistic model.

In the “original” hydra group, the effect of strain on survival time was significant (p<0.01, Fig. 2.1A). All individuals of the Nile, Carolina and Connecticut strains died within 10 hours after one hour of heat-shock at 35°C, while some individuals from the VWR and 1695C strains persisted for more than 24 hours. Most hydra lost the ability to move, contract or stick to the substrate immediately after the treatment. 33

Removing algae from the host (“aposymbiotic” group) increased thermal tolerance variation among hydra strains (p<0.01, Fig. 2.1B). Few hydra in the VWR and 1695C strains had died by the end of the observation period, and a few GBR01b hydra persisted until the end of observation, whereas the Nile, Carolina, and

Connecticut strains died out within a couple of hours after the treatment.

Novel algae-hydra associations (“novel” group) also varied in their thermal tolerances (p<0.01, Fig. 2.1C). The hydra strain with the non-hydra derived NC64A strain (from Paramecium bursaria) had intermediate tolerance. Hydra with the

Carolina algal strain had the least thermal tolerance as no polyp with this strain persisted for more than 24 hours. Hydra associated with Nile and Connecticut algal strains did not show any signs of disintegration. These two strains were in good condition, being responsive to physical stimulus after the 5-day observation period.

Only one hydra associated with the 1695C algae, its native algae, died. Hydra strains associated with algae from NC64A, GBR01b, and VWR strains experienced population declines initially, but stabilized after 96 hours.

The survival curves from the control group were in the same order as that of original hydra and aposymbiotic hydra (Fig. 2.1D), with a significant strain effect

(p<0.01). 34

Figure 2.2: Relationships between green hydra thermal tolerance and host/endosymbiont effects. Survival ranks of each strain from aposymbiotic, novel, and control groups plotted against those from original group. A lower number indicates higher survival.

Both aposymbiotic and control groups had the same rank order of Sun’s scores as the original hydra with VWR, 1695C, GBR01b, Nile, Connecticut, and

Carolina, from the most heat resistant to the least (Fig. 2.2). In the novel hydra group, Carolina was still the least resistant as in the three other groups. However, the remaining strains showed the opposite pattern. Hydra associated with algae from Nile and Connecticut had the highest survival, while in the original group they were not tolerant. VWR, which had the highest tolerance in original group, had the second lowest survival as algae in the novel background (Fig. 2.2). 35

2.3.3. Thermal Tolerance in Symbiotic and Aposymbiotic Hydra

Algal impacts were strain specific. For VWR, 1695C, and GBR01b strains, aposymbiotic individuals had higher tolerance than symbiotic ones (apo. vs. original or control: all p<0.001), and for the rest of the strains no difference was detected between symbiotic and aposymbiotic groups (0.03

Carolina p=0.010).

2.3.4. Algae Thermal Tolerance

Figure 2.3: NC64A algae chlorophyll Optical Density (OD) values following one-hour heat shock at 35°C.

36

Algae remained green after the heat shock treatment, without observable changes in color or signs of lysis. The OD value of NC64A strain almost tripled after

6 days, with no sharp decline during this period (Fig. 2.3).

2.4. Discussion

Green hydra is an appropriate model to study holobiont stress tolerance because we confirmed variation in holobiont thermal tolerance influenced by both the host and endosymbiont. By employing a bleaching and re-association approach, we were able to identify relative contributions of hosts and symbionts to holobiont stress tolerance. To our knowledge, this is the first experiment with a freshwater species to test host and endosymbiont specific contributions to holobiont stress tolerance.

2.4.1. Relative Contribution of the Host and the Endosymbiont to Hydra

Thermal Tolerance

Our results suggest that the host, rather than the symbiont, plays a stronger role in determining green hydra thermal tolerance. Variation in the thermal tolerance in the aposymbiotic (only hydra) and novel hydra groups (variety of algae in a common hydra host) reveal both host and algae affect holobiont tolerance.

However, it was the aposymbiotic hydra groups, not the novel ones, that had similar relative thermal tolerances as the original hydra groups. This strongly supports the hypothesis that the host plays an important role in the holobiont stress tolerance, something has proven difficult to directly test in corals due to their usually obligate 37 symbiotic status [175, 189]. Indirect evidence was provided by McClanahan et al.’s study [190] as they found some coral taxa were more likely to be bleached even when they occurred in different regions that varied in local environment factors. In addition, different corals hosting the same clade of Symbiodinium may not have similar susceptibility to bleaching [6]. Deriving aposymbiotic hydra from original symbiotic hydra within the same species allowed us to directly detect variation in host susceptibility to thermal stress, and how it is related to holobiont heat resistance.

In addition, comparison between thermal tolerances of the aposymbiotic and original hydra groups suggests algae impacts on holobiont thermal tolerance is either negative or neutral. In other words, having endosymbionts did not help hydra tolerate heat stress, but rather hindered their survival in those stressful conditions.

We found that bleaching significantly promoted survival of 1695C, VWR, and

GBR01b strains (the more thermal tolerant ones) while no significant impacts on the other strains (the less thermal tolerant ones) were detected. Re-associating hydra with their original algae also typically decreased survival of these tolerant strains. This is opposite to the finding that endosymbiotic algae improved

Paramecium bursaria thermal tolerance [54]. Studies on other cnidarians, however, provide evidence that endosymbionts can induce apoptosis and necrosis in situ during stress, while no signs of cell death were detected in host tissue free of symbionts [51, 53]. One explanation is damaged photosynthetic systems in the endosymbiont cause elevated oxidative stress in the host, which then can lead to degradation of the holobionts [33, 43]. Such high thermal sensitivity of 38 endosymbionts seems to be common as it has been detected in other endosymbiotic species [8, 191].

2.4.2. Adaptation Capability of Endosymbiotic Species from the

Hologenome Perspective

By showing that the host plays a major role in holobiont tolerance and the symbiont does not increase tolerance, our results indicate that holobionts such as green hydra may fail to adapt to stressful environments simply by swapping endosymbionts as suggested by hologenome theory [3]. We confirmed that tolerance conferred to the holobiont by algae is strain specific, as has been shown for endosymbionts in other species [9, 17, 93]. However, the non-positive effect on hydra thermal tolerance from the presence of algae indicates the upper tolerance limit is determined by the host, and the algae determines how close the holobiont can get to this limit. Thus, swapping symbionts may not allow the host to adapt to rising temperature if it is beyond the host threshold [3, 48]. Nevertheless, potential partners are not only limited to endosymbionts that coevolved with hydra, but may also include other symbiotic algae. Indeed, hydras in our experiment were able to form stable endosymbioses with algae from the protist P. bursaria, and showed moderate thermal tolerance. Algal symbionts in both green hydra and P. bursaria belong to the Chlorella clade, that includes symbiotic and non-symbiotic algae [192,

193]. Other endosymbiotic species may also be generalists with the potential to associate with a variety of endosymbionts, suggesting a larger partner reservoir than typically thought is available to enable them to persist in disturbances given 39 the stress is not beyond the host’s limit. Overall, our results suggest that from a hologenome perspective, adaptation of the host rather than the symbiont would have a larger effect on the holobiont thermal stress phenotype. In addition, they suggest that migration of pre-adapted hosts may have a larger impact on local holobiont tolerance than migration of pre-adapted endosymbionts.

Although not found here for green hydra, positive endosymbiont effects on host tolerance could allow a holobiont to persist in a deteriorating environment in which it could not otherwise survive. In that scenario, the baseline of holobiont tolerance is determined by the host, which may be increased by endosymbionts [17,

177]. Then endosymbiont swapping could be effective in promoting host survival when stress is beyond the host tolerance. However, such endosymbiont effects could be context dependent. For example, while algae apparently damage hosts during heat stress, they can produce mycosporine-like amino acids that protect the host from ultraviolet radiation [57, 194]. So no general conclusion can be made on whether endosymbiont swapping is effective for holobiont adaptation among different taxa, but at least swapping likely allows hosts to respond rapidly to environmental fluctuations through either bringing holobiont tolerance closer to host limits or increasing it further beyond the limits of the host alone.

Moreover, our survival data indicate selection strength is stronger on the holobiont than on either party individually, meaning endosymbiosis could turn into an evolutionary trap for the host and endosymbiotic species under changing climate.

We found low mortality for aposymbiotic hydra relative to the symbiotic hydra after 40 the heat shock treatment. In addition, algae growing independently of hydra hosts not only survived after the treatment, but also were able to reproduce days after the heat shock. Thus, both the host and the symbiont appeared to have higher likelihoods of surviving short-term stress when they were not associated with each other and could become locally extinct when they are associated. Endosymbiosis could then jeopardize survival when ambient conditions change. Since algae may be able to escape via ejection and hydra can eliminate their algae and become bleached, it is also possible this endosymbiotic relationship may become a looser association, perhaps even two independently living species [183, 195]. This may partially explain why symbiosis loss occurs in some species over evolutionary time scales

[196, 197] including some lineages of Hydra vulgaris [198].

Strong selection on the holobiont could reflect interactions between the host and symbiont. For instance, the close contact between the two parties could trap detrimental by-products which otherwise might not harm either alone. For instance, the hydra host alone may not experience a high concentration of reactive oxygen species (ROS), because a large portion is generated by the algae [53]. On the other hand, the symbiont itself could be tolerant to the ROS produced, and may be able to release it into the environment when growing alone [199, 200]. However, when algae are present in the host, the ROS could be trapped and induce host cell necrosis, which then, in turn, harms the algae [33, 43]. However, there is some evidence that

H. viridissima has evolved higher rates of ROS tolerance compared to non- endosymbiotic hydra groups [198]. The rapid increase in NC64A algae after heat shock also suggests the symbiont may more readily recover from destructive events, 41 perhaps due to their larger population sizes, simpler physiology, shorter generation times, and higher reproduction rates compared with their hosts.

2.4.3. Partner Selection in Endosymbiosis Establishment

The contrast in relative thermal tolerances between aposymbiotic hydra vs. their original algal strains in the novel hydra indicates more resistant hydra strains tend to be associated with symbionts that convey lower tolerance, and vice versa.

Bhagooli and Hidaka [201] discovered a similar pattern in dinoflagellate-coral symbiosis, that the most tolerant species in the field contained the most stress sensitive zooxanthellae. This seems to be conflict with the common assumption that tolerant symbionts are more likely to be found in harsh environments [202]. We suggest that this may be an issue of scale. On a biogeographic scale, tolerant symbionts may be more common in stressful environments but at smaller scales, less tolerant symbionts may be more likely to be associated with tolerant hosts.

Partner selection can be summarized as a two-step process. First, hosts and symbionts need to pass environmental filters to be locally available. Second, assuming the host has the dominant role in determining holobiont stress tolerance, a highly tolerant host can still survive with endosymbionts that convey weaker tolerance but a less tolerant host needs endosymbionts that convey more tolerance.

In this scenario, a tolerant host may benefit from hosting a less tolerant but less costly algal strain but a less tolerant host may only be able to persist with a strain that allows greater tolerance but at a potential fitness cost. Consistent with this, corals hosting thermally tolerant zooxanthellae grow slower than those hosting less 42 tolerant strains [203, 204]. Thus the most robust holobiont may not simply be the best host and the best endosymbiont because of other potential costs associated with hosting highly tolerant endosymbionts.

2.5. Conclusions

Our study on green hydra provides direct evidence that holobiont thermal tolerance depends on both the host and endosymbiont together. However, hydra traits were more important than algal traits, which suggests that host traits should be given more attention in future endosymbiosis studies. In addition, the lack of a positive effect of algae on hydra thermal tolerance indicates that swapping endosymbionts may not help some photosynthetic holobionts adapt to rapidly changing thermal environments. Indeed, at the extreme, having endosymbionts may transition from beneficial under benign conditions to harmful in a stressful, variable environment in which limits of tolerance are more important. At the same time, the relatively volatile thermal characteristics of small fresh water bodies compared with oceans may further destabilize photosynthetic endosymbiosis in these ecosystems but this possibility has not been explored.

43

Chapter 3

Symbiont mutagenesis and characterization as a potential method to mitigate climate change impacts on holobionts: a study on green hydra Hydra viridissima

Abstract

It is proposed that stress sensitive holobionts such as corals have to rely on their symbionts to adapt to climate change. Studies have revealed new symbiont acquisition or symbiont frequencies change can improve holobiont stress tolerance, however, whether symbiont mutations can be induced and selected to alleviate holobiont stress has not been demonstrated. Using green hydra (Hydra viridissima) and symbiotic algae (Chlorella variabilis NC64A), we found symbiont mutagenesis in vitro altered their UV-B resistance as well as that of holobionts receiving mutated algae. Hydra UV-B tolerance was positively correlated to that of the algae they were hosting. However, chronic low-level UV-B selection decreased algal resistance to acute high-level UV-B, and did not affect UV-B resistance of holobionts. Our results 44 suggest symbiont mutagenesis and trait-based identification may be more effective than assisted evolution in holobiont phenotype alteration, and it highlights the need to characterize symbiont traits in vitro that are correlated to stress tolerance they can confer for future tolerant symbiont identification. Symbiont mutagenesis and characterization potentially provides a new approach to enhance holobiont stress tolerance, which may have agricultural, forestry and conservation applications in a global climate change context.

3.1. Introduction

Discoveries of new functions provided by symbiotic microbes to their hosts have caused researchers to reconsider the significance of symbiosis in ecology and evolution, especially in the context of climate change [3, 205]. It has become clear that endosymbionts not only benefit host species by providing nutrients, protection, and energy [1, 73, 149, 206–208], but also alter host physiology and biochemistry, modifying their development and secondary chemical metabolism, and thus their stress tolerance [13, 209]. Because both the symbiont and the host determine the holobiont phenotype, Zilber-Rosenberg and Rosenberg [2] developed the hologenome theory which proposes that the holobiont should be considered as the unit of selection, and genetic changes in either party help to maintain endosymbiosis stability under stress.

Stress tolerance of holobionts, which is critical for endosymbiosis stability, is a concern in changing environments (e.g., climate change) because malfunction in 45 either the host or the symbiont may lead to endosymbiosis breakdown [169]. The positive, intimate relationship between the two parties may lead to greater vulnerability of symbiont-bearing species under climate stress compared to non- symbiotic species. Some recent studies, however, demonstrated that endosymbionts vary in their effects on holobiont tolerance which could include increasing or decreasing host stress tolerance [23, 210]. For instance, habitat stress tolerance in some plants is greatly influenced by associated symbionts [17, 22]. Given that the symbiont may play a significant role in holobiont stress tolerance, the hologenome theory suggests symbiont-bearing species may be able to respond to environmental change quickly through changing the types or relative frequencies of their symbionts [3]. This could allow rapid “adaptation” of slow-growing symbiont- bearing species via changes in symbionts, which otherwise may not be able to survive environmental changes solely through evolution of the host [47, 117]. This brought in a new perspective on modifying holobionts’ stress tolerance, which we can manipulate the symbiont rather than the host in the lab to create stress-resilient holobionts such as corals [211, 212].

It has been proposed that symbiont genomes in holobionts can shift rapidly in three ways: rare strain amplification, novel strain acquisition, and interspecific horizontal gene transfer [3]. There is evidence supporting each of these scenarios.

Studies of corals have shown switching to high thermal tolerance algae (novel strain acquisition) could increase host thermal tolerance, and the proportion of tolerant strains increased (rare strain amplification) after corals experienced heat stress [92,

95, 103]. Similarly, plants hosting different endophyte strains varied in their abiotic 46 stress tolerance [22] which supports a role for symbiont traits in holobiont stress tolerance. Pinto-Carbó et al. [107] discovered substantial horizontal gene transfer between closely related symbiotic bacteria in plants, which could facilitate their evolution. Although horizontal transmission in aphids is not as common as in corals or plants, aphids maintain symbiont polymorphisms that contribute to their success in various environments [9, 213]. Together these studies support the hypothesis that holobionts are able to quickly improve their stress tolerance simply through changing symbionts, and raise the potential of employing novel microbes to mitigate negative effects of climate change on symbiont-bearing species [22, 48]. However, these novel associations could be unstable due to high specificity of associations or symbiont-bearing species may not be able to acquire suitable symbionts due to a low abundance or diversity of tolerant symbionts in the local environment [214,

215].

One poorly investigated but promising alternative approach to increase holobiont stress tolerance relies on symbiont evolution, which can be actively intervened in the lab [212]. Their shorter generation time, higher variability, and larger populations make the symbiont a better target for directed selection as they likely can evolve faster than the host [216]. For example, the doubling time of symbiodinians is typically between days to months, but up to years for their coral hosts, and so it has been suggested that symbiodinians provide corals opportunities to persist at future elevated temperatures as more variation could be accumulated in the symbiont [23, 93, 122]. Chakravarti et al. [127] succeeded in selecting for more thermally tolerant symbionts, but failed to detect any improvements in coral 47 thermal tolerance when the algae were inoculated into hosts. Because interests of hosts and symbionts may conflict under stress, symbiont strains that have higher growth rates under stressful conditions may not confer higher stress tolerance to hosts [217]. For instance, choosing strains that have lower physiological responses to stress (such as production of reactive oxygen species) may be a better way to obtain symbionts that could improve holobiont tolerance, although the symbionts may slow the growth of holobionts under normal conditions [203], and not compete well with other strains [91]. Since standing genetic variation in the symbiont might not be enough for their adaptation to occurring stress in time [211], we decided to combine selection with mutagenesis. Researchers have succeeded in selecting for non-symbiotic, free living algal strains with specific traits via random mutagenesis

[218, 219], and so mutagenesis may potentially be used to generate genetic variability in algal symbionts, from which strains could be selected to improve holobiont stress tolerance. Ideally, it would be possible to predict holobiont phenotypes from symbiont phenotypes in order to rapidly screen a large number of candidate symbiont strains.

To explore whether we could alter holobionts’ stress tolerance by symbiont mutagenesis and selection, we used green hydra (Hydra viridissima) and endosymbiotic algae (Chlorella variabilis NC64A) as our model system [149]. We used UV-C (200-280nm) to induce mutations in the algae. We used UV-B (300-

315nm) as the selection factor in our experiments because of its detrimental effects on aquatic invertebrates [220], which can be attenuated by endosymbiotic algae via shading or mycosporine-like amino acids (MAAs) [57, 221, 222]. In this experiment, 48 we addressed the following questions: 1) Does mutagenesis and/or selection alter algae stress tolerance? 2) Does symbiont mutagenesis and/or selection change holobiont stress tolerance? 3) How are symbiont and holobiont stress tolerances related? By answering these questions, we will be able to verify the feasibility of rapid holobiont phenotype alteration through symbiont mutagenesis and selection, without the need of cultivating holobionts during the strain-identifying process

[223].

3.2. Materials and Methods

3.2.1. Study species and culture conditions

Endosymbiotic algae (Chlorella variabilis strain NC64A) used in the experiment were provided by Dr. David Dunigan (University of Nebraska-Lincoln,

USA). This algal strain was isolated from Paramecium bursaria and cultivated in

Modified Bold's Basal Medium (MBBM) [182]. We cultured algae in a constant 20°C walk-in culture room with a 25-watt, 55.88 cm-long fluorescent light placed 40 cm above the cultures for illumination, providing a 12:12 hour light-dark cycle.

Aposymbiotic (hydra without algae) green hydra (Hydra viridissima strain

1695C) were provided by Dr. Daniel Martinez (Pomona College, CA, USA). This hydra strain was collected in Chile in its symbiotic state, then bleached and maintained in the lab for generations. We kept hydra in petri dishes in a constant

18°C room. We cultured them in hydra medium [181] changed weekly and fed them 49 brine shrimp (Brine Shrimp Direct, Ogden, Utah, USA) two times a week. Hydra received the same illumination as the algae.

The experiment involved two main parts (summarized in Fig. 3.1).

a. Algae mutagenesis and selection

Long-term UV-C UV-B Exposure Mutated

1.Mutagenesis 2.Selection Original algae Non-mutated No UV-B

control (Non-selected Control)

Selected

Short-term UV-B Exposure

3.UV-B Resistance Non-selected Test No UV-B

b. Algae-hydra reassociation and resistance test

Selected Non-selected

4.Hydra 5.Hydra UV-B Reassociation Resistance test Data Analysis

Algae from step 2

Figure 3.1: Experiment flowchart of (A) algae mutagenesis and selection, (B) hydra-algae reassociation. The goal was to test whether we could alter symbionts’ and holobionts’ stress tolerance via symbiont mutagenesis and/or selection. Symbiotic algae NC64A was mutated with UV-C (step 1) and selected with UV-B (step 2). Both selected and non-selected populations were tested for UV-B resistance (step 3) and inoculated into hydra (step 4). Hydra were than tested for their UV-B resistance (step 5) which allowed the stress tolerances of symbionts and holobionts to be compared. Light fill indicates algae that have not been mutated, dark fill indicates mutated algae, solid lines 50 indicate algae that have not undergone selection and dashed lines indicate algae that have undergone selection.

3.2.2. Algae mutation and selection

We followed a mutation-selection process on algae to determine if we could change algal UV-B resistance, and therefore hydra UV-B resistance [224]. We collected algae at log growth phase (105– 106 cells/ml) and combined them to get homogenized algae suspensions for mutagenesis [225]. We filled 10 cm-diameter petri dishes with 15 ml of this suspension and subjected them to 0.7 mw/cm2 UV-C exposure, for periods of 10, 15, 20, 25, 30, 35, 40, 45, 50, or 60 minutes in a dark room and kept two additional groups unexposed. After the exposure, we immediately transferred algae of each dish into a 50ml test tube and placed them in a dark chamber to prevent photoreactivation [224, 225]. After 24 hours, we transferred the tubes to the culture room, and left them there for three weeks until algae recovered or died. We also placed 20 polyps of symbiotic green hydra that hosted the original NC64A algae under the same UV-C strength for 30 minutes to check if the holobiont could survive the stress the unassociated algae received. We used algal cultures that recovered from the UV-C treatment for further experiments.

Cultures A and B were non-mutated algae; cultures C through I were mutated algae from recovered tubes. We divided each culture into a non-selected (control) population and at least one UV-B treatment population (one for all but culture F which had three) and inoculated them into continuous culture independently. Each 51 culture was an aerated 250 ml glass side arm flask containing 200 ml of MBBM.

Medium was added at a rate of 100 ml/day (Carter Cassette Manostat Digital Pump,

Anderson Process, Brookfield, WI USA) and discharged from the outlet. Cultures were kept at 18°C with the same illumination as described above. The UV-B treatment group received an additional 0.15 w/m2 UV-B irradiance for 12 hours daily. After two months, we collected algae into test tubes and cultured them for approximately another eight generations before UV-B resistance tests to control for acclimation effects [226].

3.2.3. Algae UV-B resistance tests

We centrifuged each test tube with algae, removed the supernatant, and re- suspended algae with deionized water, three times, to get rid of nutrients for algae growth. We divided each population (culture and selection treatment combination

[selected and non-selected]) into three groups: 0hr time point group, 48hr time point control group, and 48hr time point short-term UV-B treated group. There were three replicates for the 0hr time point group and the 48hr time point control group respectively (2 ml aliquots in 10 ml glass test tubes), and at least 12 replicates for the 48hr time point short-term UV-B treated group. We placed the 48hr time point control group at 18°C with the same illumination as described above for 48 hours; the 48hr time point short-term UV-B treated group received the same illumination plus 0.2 w/m2 UV-B. For each population, we centrifuged the tubes, removed the supernatant, added 1 ml methanol, sealed the tube with parafilm, sonicated it for 1 minute (40 kHz), kept it in the dark at 5°C overnight, and 52 measured optical density at 663 nm (OD0hrs). The OD reading was used as an estimate of algae density because density is linearly proportional to OD [227]. We used a higher intensity for the tests of algae UV-B tolerance because the goal of the selection experiment was to generate differences in growth and survival while allowing long-term cultures but the goal of the assays of tolerance phase was to have UV-B levels intense enough to quantify UV-B tolerance for a range of algae and holobionts.

3.2.4. Regreened hydra UV-B tolerance test

For each population (culture and selection treatment combination [selected and non-selected]), we microinjected algae into aposymbiotic hydra. We picked and cultured hydra that turned green in hydra medium. We placed 25 to 75 re-greened non-budding polyps of similar size from each algal culture and selection treatment at 18°C with the same illumination as described above plus 0.1 w/m2 UV-B, and tracked the population sizes over 48 hours.

3.2.5. Statistical analyses

We performed a pair of ANOVAs (proc mixed, SAS 9.4) to determine the dependence of the change in OD for each population (ln[OD48hrs/OD0hrs]) on selection, culture, selection×culture and population(selection×culture) in control or

UV-B test conditions. We used adjusted means partial difference tests to discriminate among means for significant factors. To estimate tolerance to UV-B, for each population, we randomly assigned a UV-B test pair to each control test pair to 53 create 5000 pseudo-datasets and calculated relative change in OD over 48 hours (ln

([ODUVB,t=48hrs / ODUVB,t=0hrs] / ([ODcon,t=48hrs / ODcon,t=0hrs])). We calculated quartiles and 95% confidence intervals (CI) from these 5000 randomizations. We also calculated the average non-selected and selected algal UV-B resistances

(excluding culture E that only had selected algae survive). We considered non- selected and selected populations to differ in their UV-B resistances when their 95%

CIs did not overlap.

We used another ANOVA (proc glimmix, binomial distribution, logit link) to test whether hydra survival with UV-B exposure depended on selection and selection×time. We did not include cultures that did not have both non-selected and selected algae associated with hydra. We used survival analysis (proc phreg, Cox proportional hazard model) to test how hydra mortality under UV-B depended on algal culture and selection.

To test how algal survival under UV-B test conditions was related to hydra survival under UV-B test conditions when associated with those algae, we performed a correlation (proc corr) between the estimates of change in OD of algal populations under UV-B test conditions as the predictor and the Cox proportional hazard model estimates for hydra as the response. We repeated this correlation only including cultures that had both non-selected and selected populations associated with hydra. 54

3.3. Results

We ended up with surviving algal cultures that had been exposed to 0 (N=2),

15 (N=1), 20 (N=1), 30 (N=4), or 35 minutes (N=1). In addition, some algal populations were lost later during the experiment (non-selected culture E population) and some surviving populations were not associated with hydra (non- selected culture C, D, and I populations). All the hydra died within one day after UV-

C exposure.

The change in OD for algae in control test conditions did not depend on selection (F1,29=1.0, P=0.3367), culture (F7,29=1.6, P=0.1698), selection×culture

(F7,29=1.5, P=0.1992), or population(selection×culture) (F2,29=0.2, P=0.8215; Fig.

3.2A). In UV-B test conditions, selected algae had larger reductions in OD on average

(F1,164=13.4, P<0.0001) and reductions depended on culture (F7,164=13.3, P<0.0001).

But selection only significantly reduced OD for some populations (selection×culture,

F7,164=1.9, P=0.0777; population(selection×culture), F2,164=3.1, P=0.0463; Fig. 3.2B).

UV-B resistance of algae was lower with selection on average and for three of the selected cultures (Fig. 3.2C). 55

Figure 3.2: The change in OD at 663 nm (ln [ODt=48hrs / ODt=0hrs]) for different non-selected (dark symbols or lines) or selected (light symbols or lines) populations of algae from different cultures in (A) control and (B) UV-B test conditions. Means ± se. Means with the same letters did not differ in post-hoc tests. * indicates p<0.05. (C) Algal tolerance to UV-B in test conditions (ln

([ODUVB,t=48hrs / ODUVB,t=0hrs] / ([ODcon,t=48hrs / ODcon,t=0hrs])) estimated by randomization. Thick bars indicate quartiles. Thin lines indicate the 95% CI. * indicates 95% CI that do not overlap for non-selected vs. selected algal populations from the same culture. A,B = 0 min UV-C (non-mutated), C = 15 min, D = 20 min, E,F,G,H= 30 min, I = 35 min. Overall indicates the average of cultures with both selected and non-selected populations (excludes E). 56

Hydra survival in UV-B test conditions varied among cultures (F6,121=8.6,

P<0.0001) but did not depend on algal selection (F1,121=2.3, P=0.1358) or their interaction (F4,121=1.8, P=0.1385) (Fig. 3.3).

100

80 3

60 1 40 2 A-non A-selected 20 B-non F-non B-selected F-selected 0 100

Survival (%) Survival 80

60

40 G-non C-selected G-selected 20 D-selected H-non E-selected H-selected I-selected 0 25 30 35 40 25 30 35 40 HoursDays

Figure 3.3: The survival of hydra associated with different non-selected (dark symbols or lines) or selected (light symbols or lines) populations of algae. A,B = 0 min UV-C (non-mutated), C = 15 min, D = 20 min, E,F,G,H= 30 min, I = 35 min. 57

Algal UV-B resistance and hydra UV-B resistance were significantly positively correlated (r=+0.52, P<0.05) whether all populations were included or only algae cultures with both selected and non-selected types (Fig. 3.4).

Figure 3.4: The correlation (line) between algal change in OD in UV-B (Fig. 3.2B) and Cox survival coefficient for different non-selected (dark circles) or selected (light squares) populations of algae. A,B = 0 min UV-C (non-mutated), C = 15 min, D = 20 min, E,F,G,H= 30 min, I = 35 min. Non-selected culture A set at the origin. 58

3.4. Discussion

The hologenome theory proposes that rapid genetic changes in symbionts’ genes may allow holobionts to adapt to new environments quickly, which implies artificial modification of symbionts’ genes could be employed to develop holobionts with increased stress tolerance [3]. Targeting the symbiont rather than the holobiont for selecting stress-tolerant holobiont is likely to be more efficient because of their shorter generation time, larger quantities that can be collected, ease of cultivation, and possibly more resilience to mutagenesis [47, 216, 228]. Our results explicitly demonstrated that we can alter a holobiont’s stress tolerance by mutating their associated symbionts, and we revealed a positive correlation between the symbiont’s and the holobiont’s stress tolerance. By following the mutation-selection process that had been conducted on free-living microbes [218,

219, 224], we successfully created symbiotic algal populations that varied in their

UV-B resistance, which could be conferred to hosts. In addition, we can predict the impacts of symbionts on holobiont stress tolerance before we inoculate them.

Interestingly, we found it was mutagenesis but not selection on symbionts that affected holobiont stress tolerance. The results support the hologenome theory, and suggest symbiont mutagenesis combined with identification of symbionts with particular traits could be an efficient approach to acquire desired holobiont phenotypes. Because we could cultivate the symbionts in vitro, it was easy to generate such variations and identify algae that confer higher resistance to hosts. 59

Our experiment indicates that random mutations can be induced in symbionts independent of hosts and influence holobiont phenotypes, suggesting we can use isolatable symbionts that subsequently can be associated with hosts to improve holobionts’ stress tolerances [229]. When compared with the non-mutated algal cultures, mutated algal cultures exhibited significantly different UV-B resistance, as did green hydra associated with mutated and non-mutated algae. This provides new insights into holobiont trait alteration, because most past research tested various naturally occurring strains for their impacts on holobiont traits, and mutagenesis was only applied to identify genes that are responsible for parasitism vs. mutualism [230, 231]. For example, Rodriguez et al. [22] found naturally occurring fungal strains differ in the stress tolerance conferred to host plants.

Similarly, coral thermal tolerance is also influenced by the types of symbionts they host [93]. In a previous work, different naturally occurring algal strains were associated with different naturally occurring hydra strains, and it was found that holobiont thermal tolerance varied across algae strains, with NC64A conferring intermediate tolerance [232]. It is possible that the choice of this non-native algal strain influenced our results here because other researchers have shown that this can have an impact on some holobiont traits [149] . Here we demonstrated that in addition to employing naturally occurring symbionts, we can further generate variations based on a known beneficial symbiont strain and choosing those that confer greater stress tolerance to hosts. Perhaps the next logical extension is to identify, or also maybe modify, functional genes in symbionts that contribute to 60 improved holobiont stress tolerance or production, such as heat shock genes or enhanced nitrogen-fixing genes.

In addition, our results also indicated symbionts are more suitable for a mutation-selection process than holobionts, because we discovered free-living symbiotic algae but not holobionts survived mutagenesis. We found all symbiotic hydra died after a 30-minute UV-C exposure; in contrast, free-living algae recovered from the same exposure conditions. The greater chances of mutation due to higher mutagen dose, plus larger symbiont population sizes that could receive mutagenesis, suggest the potential of generating induced mutations is higher in the symbiont than in the host [233]. These mutations may be especially likely to generate novel traits in symbionts because their genome size is usually smaller and more compact than their free-living relatives, retaining mostly functionally critical genes [234, 235]. Symbionts significantly contribute to holobiont functional traits, so changes in these genes could easily alter symbionts’ phenotypes, and thus holobionts’ phenotypes [66, 236, 237]. Modifying cultivable symbionts is not only feasible but also seems to be more practical than modifying the host or the holobiont directly to acquire desired holobiont phenotypes.

While we found mutagenesis altered algal UV-B resistance, we failed to detect any UV-B resistance improvement due to selection. In fact, some algae populations showed lower UV-B resistance after they experienced long-term UV-B exposure, indicating stress induced damage rather than adaptive responses in these algae. In contrast, some previous studies detected either positive or no effect of 61 long-term selection on microorganisms’ fitness under stress. For example, Lohbeck et al. [226] demonstrated adaptive evolution of the coccolithophore alga Emiliania huxleyi to elevated CO2 in 500 generations, and Huertas et al. [238] found 12 algal strains of a variety of species were able to improve their thermal tolerance after 8 to over 100 generations under elevated temperature. Thermal adaptation was also observed in free-living algal coral symbionts, as their fitness was higher at elevated temperature after 80 generations of selection [127]. Yet a longer selection experiment on the green algae Chlamydomonas at elevated CO2 for over 1000 generations failed to detect any adaptive responses, even though they found some new traits occurred, probably as the result of accumulated neutral mutations [239].

Sexual reproduction has not been observed in Chlorella so our study almost certainly involved selection on asexually reproducing algae, but other studies included algae that can undergo sexual reproduction [240, 241], which could help to explain the range of selection responses reported in the literature but not the negative effect of selection in our study. In addition, negative carry over effects might occur, in which long term exposure to stress in ancestors decreases offspring fitness [242]. An alternative explanation could be that impacts of lower magnitude, chronic UV-B and higher magnitude, acute UV-B differed (because of different objectives for the selection vs. assay stages), so our selection process did not select for algae that had relevant tolerance in our assays. Thus we propose for future symbiont selection experiments that the selection criteria and final testing criteria should be the same, especially if the mechanism underlying their effect on host, and in turn, holobiont traits are unknown. 62

In newly associated hydra, we detected effects of symbiont mutagenesis but not symbiont selection on holobionts’ UV-B resistance. Hydra that received different populations of algae had dissimilar UV-B resistance, and the positive correlation between algal UV-B resistance and symbiotic hydra UV-B resistance suggests algae are able to confer their UV-B resistance to hosts. In addition, algae from the same culture conferred similar UV-B tolerance to hydra no matter if they were selected or not. This resembles the results of some previous studies. For example, Giauque et al.

[243] found naturally occurring stress tolerant endophytic fungi improved plant fitness under stress; and Chakravarti et al. [127] observed higher thermal tolerance for selected free-living algal strains but the selection effect disappeared when algae were associated with coral hosts. Because both biochemical and physiological attributes affect algal UV-B resistance, certain algal strains that differ when free- living may not vary within the host as forming endosymbiosis changes their morphology or their spatial distribution [244]. This might also explain why

Chakravarti et al. [127] failed to detect coral thermal tolerance improvement after they inoculated corals with symbionts having highest growth rates at elevated temperature. Our results together with those from previous studies suggest symbiont selection might not be effective in improving holobiont stress tolerance, particularly if tolerance-conferring traits of symbionts are not clearly identified

[212].

Indeed, our results not only show that experimentally induced variations in symbionts could generate various holobiont phenotypes, but also suggest stress tolerant symbionts confer greater tolerance to hosts. Depending on the species and 63 traits of interest, predicting the holobiont phenotype based on the symbiont phenotype is possible. For instance, experimentally replacing naturally occurring bacterial symbionts of aphids with more heat tolerant bacteria increased aphid thermal tolerance [245]. In our case, hosting a stress tolerant symbiont population was likely to increase the holobiont fitness under stress. There are, however, symbiont traits other than stress response that can predict symbiont effects on holobiont performance. In endophyte-plant symbiosis, fungi traits related to resource use and habitat characteristics can predict up to 53% of their effects on plants [243]. Previously, Mueller and Sachs [223] proposed engineering animals and plants with microbes, but their method (“host-mediated selection”) is indirect and requires cultivating the holobionts. Here we propose direct manipulation of symbionts, which is faster and more economical efficient, because more symbionts can be screened in a shorter time. Moreover, instead of conducting selection on symbionts, generating and identifying symbionts with certain functional traits via mutagenesis could be more effective in finding symbionts that enhance holobiont- stress tolerance.

3.5. Conclusions

To the best of our knowledge, our experiment is the first to employ symbiont mutagenesis to improve holobiont stress tolerance, which highlights the ability of holobiont evolution via rapid symbiont evolution. It also reveals a novel approach that can be employed to engineer holobionts. Compared with conventional host- targeted breeding and genetic engineering, symbiont-based approaches are time- 64 efficient and transferrable (i.e., can be applied to multiple species) [246], and has been applied in agriculture using non-mutagenesis methods [247]. Because microbiomes can improve plant stress tolerance and they have been successfully applied to promote plants growth under unfavorable conditions [20, 248], we propose that symbiont mutagenesis, evaluation and inoculation could potentially be applied to achieve desired traits of these and other organisms [223] for agricultural or conservation purposes. Although there are important policy dimensions and ecological consequences (e.g., reduced biodiversity) to consider, this strategy could be applied to conservation or agriculture by creating and cultivating strains in the lab then releasing them to increase holobiont stress tolerance in the field [212].

Moreover, altered traits due to newly associated symbionts may be inherited vertically for some holobionts [98]. In our case, we found that holobiont and symbiont abiotic tolerances were positively correlated but that generation and screening of symbiont variants was a more effective approach for improving holobiont tolerance than directed host-mediated symbiont selection [223]. Having a better understanding of correlations between microbes’ functional traits and their effects within holobionts may allow assessment of numerous symbiont strains without inoculating them into the host, or even gene editing the symbionts to produce holobiont-enhancing symbiont strains. Future research should investigate the mechanisms by which symbionts affect holobiont stress tolerance in order to identify symbionts with traits that are likely to improve their fitness under stress. 65

Chapter 4

Thermal plasticity of a freshwater cnidarian holobiont: detection of trans- generational effects in asexually reproducing hosts and symbionts

This chapter has been re-formatted, modified, and reprinted from: Ye S,

Badhiwala KN, Robinson JT, Cho WH, Siemann E. 2019. Thermal plasticity of a freshwater cnidarian holobiont: detection of trans-generational effects in asexually reproducing hosts and symbionts. ISME J 13: 2058–2067.

Abstract

Understanding factors affecting the susceptibility of organisms to thermal stress is of enormous interest in light of our rapidly changing climate. When adaptation is limited, thermal acclimation and deacclimation abilities of organisms are critical for population persistence through a period of thermal stress. Holobionts 66

(hosts plus associated symbionts) are key components of various ecosystems, such as coral reefs, yet the contributions of their two partners to holobiont thermal plasticity are poorly understood. Here, we tested thermal plasticity of the freshwater cnidarian Hydra viridissima (green hydra) using individual behavior and population responses. We found that algal presence initially reduced hydra thermal tolerance. Hydra with algae (symbiotic hydra) had comparable acclimation rates, deacclimation rates, and thermal tolerance after acclimation to those without algae

(aposymbiotic hydra) but they had higher acclimation capacity. Acclimation of the host (hydra) and/or symbiont (algae) to elevated temperatures increased holobiont thermal tolerance and these effects persisted for multiple asexual generations. In addition, acclimated algae presence enhanced hydra fitness under prolonged sublethal thermal stress, especially when food was limited. Our study indicates while less intense but sublethal stress may favor symbiotic organisms by allowing them to acclimate, sudden large, potentially lethal fluctuations in climate stress likely favor aposymbiotic organisms. It also suggests that thermally stressed colonies of holobionts could disperse acclimated hosts and/or symbionts to other colonies, thereby reducing their vulnerability to climate change.

4.1. Introduction

Severe ecological and economic consequences of symbiotic species failure have raised concerns due to the ubiquitous distribution of symbioses in nature [1].

Consequently, we need to understand how mutualistic symbiotic species may respond to climate change in the long term [34, 250]. Symbiotic species are thought 67 to be more susceptible to environmental stress because the symbiosis may breakdown from malfunction in either party, plus most symbionts and some hosts reproduce asexually, where adaptation via genetic recombination is limited [1, 169,

251–253]. Thermal tolerance is a critical factor that determines symbiotic species stability under climate change, since many symbiotic species, from protists and plants to invertebrates, are poikilotherms that are sensitive to thermal conditions, especially temperature stress [254]. However, thermal tolerance of symbiotic species likely depends on characteristics of the host and/or symbiont.

According to the hologenome theory, holobiont (host plus symbiont) thermal tolerance is governed by both the symbiont and the host genes, yet impacts of their plasticity are overlooked [3]. Previous studies have revealed that both hosts and symbionts contribute to a holobiont’s stress tolerance [9, 175, 255], and different symbiont or host strains may vary in the tolerance they confer [93, 119]. Yet we are not aware of any published study that reports how thermal stress affects algal densities in green hydra. Our previous study on green hydra revealed that although symbiotic hydra did not bleach under heat stress as other marine cnidarian do, they died faster than aposymbiotic hydra, indicating being symbiotic decreased hydra thermal tolerance [232, 249, 256]. However, the long-term success of species, especially for asexually reproducing ones, under climate change is not only determined by their acute stress sensitivity but also by their plasticity [257–259].

Thermal plasticity governs the species’ potential to expand thermal tolerance under prolonged stress and it consists of two main components: (1) acclimation capacity and (2) acclimation rate [260, 261]. Acclimation capacity determines the magnitude 68 of thermal tolerance change; acclimation rate determines how fast the organism can reach this limit [262, 263]. Both are critical to species persistence under climate change. For example, species that live close to their thermal limits with little acclimation capacity are threatened by even small temperature increases [264]; species with low absolute thermal tolerance and high acclimation capacity may persist even when the ambient temperature elevates by a large amount, but they may need to acclimate quickly or they may perish [265]. Some studies suggest that symbiont acclimation could buffer holobionts during stress [118, 135], but there is a lack of direct experimental evidence. Therefore, understanding how the host and symbiont affect holobiont acclimation is critical for predicting the impacts of long- term temperature stress on symbiotic species.

To date, how symbiont presence affects holobiont acclimation is unclear, and how acclimation in the symbiont and in the host influence holobiont thermal tolerance have not been well studied. Most experiments focused on detecting variation in acclimation responses among different holobiont populations conducted reciprocal transplants [132, 133]. Although these studies revealed acclimation could occur in both the host and the symbiont, they did not further explore relative contributions of acclimation in each party to holobiont thermal tolerance, nor did they investigate the rate or the magnitude of such acclimation. For example, researchers found symbionts such as dinoflagellates and arbuscular mycorrhizae were able to acclimate to heat, but they did not test impacts of such acclimation on holobionts [135, 266]. Because symbiotic organisms are often very sensitive to heat [5, 71, 191], it has been suggested that they may be less able to 69 acclimate to thermal stress compared to aposymbiotic organisms [8]. Manipulative studies of holobiont acclimation are rarely performed and even then host and symbiont acclimation are coupled due to the difficulties in separating them [267]. In addition, past studies neglected responses of holobiont populations during thermal acclimation, instead they monitored individual physiological responses, such as transcriptome, protein content, algae density, or respiration [129, 132, 135, 260,

268]. The lack of holobiont population data could limit our ability to predict their population dynamics under global climate change so direct experiments and observations are needed.

Another critical process that shapes poikilotherms’ success in unstable temperature environments is deacclimation, especially the rate and the extent to which the organism loses gained tolerance [269]. Cold acclimated organisms such as plants could lose their resistance during transient warm periods in winter, and are subject to damage accrued from subsequent low temperatures [270, 271]. Thus a low deacclimation rate may be beneficial so the organism could retain acquired tolerance to overcome recurring stresses. In fact, studies have revealed that such acquired plasticity could even persist in both asexually and sexually reproduced offspring, which are known as trans-generational effects [259, 272, 273]. However, for symbiotic species, such trans-generational effects have not been investigated for the host and the symbiont independently, and their impacts over multiple asexual generations is not well studied [259, 274, 275]. 70

In this study, we conducted experiments on the freshwater cnidarian Hydra viridissima (green hydra) and derived aposymbiotic hydra (hydra without algae) to understand how thermal acclimation and deacclimation in the host (hydra) and/or the symbiont (algae) affect holobiont thermal tolerance. In addition, we tracked hydra motion activities during acclimation using microfluidic micro-arenas designed for hydra [276] . Hydra contract spontaneously under normal conditions, and stress could disrupt both the rhythm and magnitude of the movements [277].

Tracking hydra motions allowed us to make direct observations of the acclimation rates of symbiotic and aposymbiotic hydra, and detect algal impacts on hydra acclimation at the individual physiological level. We also reassociated acclimated hydra hosts with non-acclimated algal symbionts and vice versa, to tease apart the roles of symbiont and host in holobiont thermal acclimation and deacclimation.

Although green hydra is a simple cnidarian model with low intra-host symbiont diversity in contrast to more complex symbiotic organisms such as corals, our work could have general implications for other symbiotic species [278–282].

4.2. Materials and Methods

4.2.1. Focal organisms

Hydra viridissima is a freshwater invertebrate that hosts symbiotic green algae (Chlorella sp.) in its body [283]. The host relies on the symbiont for carbohydrates, while the symbiont relies on the host for nitrogen compounds [178,

179]. However, aposymbiotic hydra (those that have had their symbionts 71 experimentally eliminated) can live independently if given enough food and can be reassociated with algae [180, 183].

Hydra used in our experiment were strain 1695C acquired from Dr. Daniel

Martinez at Pomona College, CA, and originally cultured in an 18°C (optimal temperature) walk-in room. For our experiments, these hydra were subjected to different thermal regimes to acquire five different acclimation states. 1) Non- acclimated (control) hydra continued to be kept at 18°C. 2) Acclimating hydra were those transferred to a 30 ±1 °C (sublethal heat stress) walk-in room and maintained at the elevated temperature for no more than one month, 3) Acclimated hydra were those that had been cultured at 30°C for two months. 4) Deacclimating hydra were previously acclimated hydra transferred back to 18°C and maintained at this optimal temperature for no more than one month. 5) Deacclimated hydra were previously acclimated hydra transferred back to 18°C for two months. All hydra were illuminated by 3000 lux fluorescent lights with a 12:12 hour light-dark cycle in petri dishes in hydra medium [181].

4.2.2. Population Growth Rate Measurement

We measured population growth rates of symbiotic and aposymbiotic hydra in non-acclimated, acclimating, and acclimated states. For both symbiotic and aposymbiotic hydra, eight replicates containing five non-budding hydra in the non- acclimated state were fed with brine shrimp (Brine Shrimp Direct, Inc., Ogden, UT,

USA) and counted on Monday, Wednesday and Friday every week [181]. The experiment was terminated when any hydra population reached 100 polyps to 72 exclude effects of crowding. Similarly, six replicates of non-acclimated symbiotic and aposymbiotic hydra (five polyps each) were transferred to 30°C respectively, then fed and counted (as described above) to acquire population growth curves of acclimating hydra. The same process was applied to acclimated hydra.

4.2.3. Contraction Activity Monitoring

We monitored symbiotic and aposymbiotic hydra contraction frequency for the first 12 hours of acclimation. Eight symbiotic and eight aposymbiotic hydra were placed together into each of a pair of microfluidic behavioral micro-arenas (i.e.,

N=32) previously reported for constraining hydra movements in the vertical direction [276]. The microfluidic chip was fabricated using conventional microfabrication techniques and permanently bound to a glass slide. The master mold for the micro-arenas was 3D printed with 1-mm tall parallel chambers. The two chips were transferred from the 18°C culture room to a 24°C room for two hours (to avoid potential heat shock effects) before being transferred to a 30°C water bath. We took pictures of hydra at one frame per second (EOS T3 camera,

Canon USA, Melville, NY, USA). The pictures were then processed with Adobe After

Effects (Adobe Systems, San Jose, CA, USA) to count the number of full contractions for each symbiotic and aposymbiotic hydra on an hourly basis. A full contraction was defined as: hydra contracts from an elongation position that is greater than

50% of the maximum length to under 20% of the maximum length. We repeated this procedure with non-acclimated hydra at 18°C for six hours and acclimated hydra at

30°C for six hours (N=32 for each [eight symbiotic & eight aposymbiotic in two 73 chips]).

4.2.4. Thermal Tolerance

Non-acclimated, acclimated and de-acclimated hydra thermal tolerances were assessed by their persistence time after they were transferred from their cultivation temperature to a 38°C (lethal heat stress) water bath. Longer persistence time implies higher upper thermal limits. This temperature level was chosen because treatments varied at this temperature in a pilot study. The endpoint was the time hydra started to disintegrate. Twenty-four to forty non-budding polyps from each group were randomly selected for the experiment.

Acclimating symbiotic and aposymbiotic hydra thermal tolerances were assessed on day 0.5, 1, 2, 3, 5, 7, 14 and 21 after they were transferred from 18°C to

30°C. Each time, eight to ten non-budding hydra polyps were randomly picked from each of the populations, and their persistence times were measured at 38°C as described above. Thermal tolerances of deacclimating hydra were assessed in a similar way after acclimated hydra were transferred back to 18°C. During acclimation and deacclimation, hydra were fed on a weekly basis to limit budding.

To further explore how symbionts could affect hosts during long-lasting thermal stress under starvation, we kept 20 acclimated aposymbiotic hydra and 20 acclimated symbiotic hydra at 30°C for 20 days without feeding. A control group of

20 non-acclimated aposymbiotic hydra and 20 non-acclimated symbiotic hydra were kept at 18°C for 20 days without feeding. 74

To evaluate effects of acclimation by the host vs. the symbiont on holobiont thermal tolerance and their stability across asexual generations, we reassociated hydra and algae with different thermal histories, and created four types of symbiotic hydra that had previously experienced high, elevated temperature (30°C) in: 1) both the hydra and the algae, by injecting algae removed from acclimated symbiotic hydra (HA: high-temperature history algae) into acclimated aposymbiotic hydra

(HH: high-temperature history hydra). 2) only the hydra, by injecting algae removed from non-acclimated symbiotic hydra (LA: low-temperature history algae) into acclimated aposymbiotic hydra. 3) only the algae, by injecting acclimated algae into non-acclimated aposymbiotic hydra (LH: low-temperature history hydra). 4) neither the hydra or the algae by injecting non-acclimated algae into non-acclimated aposymbiotic hydra. All hydra that turned green were picked and cultured at 18°C until the algae density stabilized after 3 weeks, from which another three generations were derived [124, 284, 285]. We then placed 50 non-budding polyps of each type in beakers with hydra medium in a 37°C water bath separately. The hydra were re-located to 18°C after 70 minutes, and their survival (determined by whether completely disintegrate or not) was tracked for four days.

4.2.5. Statistical Analysis

We analyzed the dependence of population sizes (log transformed) on acclimation status (non-acclimated, acclimating, or acclimated hydra), symbiosis status and their interactions in a repeated measures ANOVA (Proc Mixed, SAS 9.4) where time was treated as a categorical fixed variable. Then we used an ANCOVA 75

(continuous time) and post-hoc tests to compare the exponential population growth rates for different combinations of acclimation and symbiosis statuses.

We examined the dependence of individual contraction rates on acclimation status (non-acclimated or acclimated), symbiosis status, and their interaction in an

ANOVA (Proc Mixed). We used additional repeated-measures ANOVAs to analyze the dependence of individual contraction rates on symbiosis status during the acclimating and acclimated stages. We used a segmented regression [286] to analyze contraction frequencies during the first 12 hours of acclimation (segmented package, R v3.4.3). This enabled us to compare the breakpoints of symbiotic and aposymbiotic hydra during acclimation, which were the times they stopped exhibiting abnormal behavior.

We examined the dependence of persistence time at 38°C on acclimation status

(acclimating or deacclimating), symbiosis status and their interaction with a repeated-measures ANOVA (Proc Mixed). We used an ANOVA to test the dependence of persistence time at 60 days on acclimation status (non-acclimated, acclimated, deacclimated), symbiosis status and their interaction (Proc Mixed).

We tested the dependence of starving hydra survival time under prolonged sublethal heat stress on symbiosis status using an ANOVA (Proc Glimmix, binomial distribution, logit link, SAS 9.4).

Finally, we performed a set of pairwise analyses to examine the survival time of different combinations of hydra and algal acclimation (Proc Lifetest, method=Kaplan-Meier, SAS 9.4). 76

4.3. Results

140 Acclimation status Symbiosis status Non-acclimated Aposymbiotic Acclimating Symbiotic 120 Acclimated

100

80

60 Population size

40

20

0 0 10 20 30 Time (days)

Figure 4.1: Growth of symbiotic hydra (green symbols and lines) and aposymbiotic hydra (black symbols and lines) at 18°C before acclimation (controls - squares), acclimating at 30°C (triangles), or acclimated at 30°C for two months (circles).

On a population level, acclimation status (F2,320=120.84, p<0.0001), symbiosis status (F1,320=94.28, p<0.0001), and their interaction (F2,320=66.66, p<0.0001) had significant effects on hydra growth rate (Fig. 4.1). Population growth 77 rates were highest for non-acclimated (especially aposymbiotic), intermediate for acclimated, and lowest for acclimating (higher for aposymbiotic) (Table 4.1).

Acclimation status Symbiosis Status Growth Rate k Before Acclimation (at 18°C) Symbiotic 0.119±0.004 b (Non-acclimated - controls) Aposymbiotic 0.137±0.004 a During Acclimation (at 30°C) Symbiotic 0.072±0.003 e (Acclimating) Aposymbiotic 0.081±0.003 d After Acclimation (at 30°C) Symbiotic 0.097±0.003 c (Acclimated) Aposymbiotic 0.089±0.007 cd

Table 4.1 Growth rates of symbiotic and aposymbiotic hydra with different acclimation and symbiosis statuses (ln[#] = a + k×days). Growth rates with the same letters were not different in post-hoc tests.

On the individual level, hydra contraction frequency was higher after they acclimated to 30°C (Time 1440hrs, Fig. 4.2) compared with that at 18°C (F1,111=5.98, p=0.0160, Time 0hrs, Fig. 4.2) but the difference did not depend on symbiosis status

(F1,111=1.13, p=0.2899). During acclimation, the contraction frequency of symbiotic and aposymbiotic hydra declined first and then recovered (Fig. 4.2). From hour 1 to

5, the contraction rate declined more rapidly for symbiotic than for aposymbiotic hydra (F1,148=14.73, p=0.0002). The contraction rate was higher for aposymbiotic hydra from hours 5 to 12 (F1,239=4.92, p=0.0275) but the contraction rates did not vary with time overall (F1,239=3.21, p=0.0746) or between types (F1,239=0.31, p=0.5813). The estimated breakpoint for aposymbiotic (4.12 ± 0.42 hours) and symbiotic (4.34 ± 0.38 hours) hydra were similar (p=0.7060). Here the breakpoint refers to a switch from declining to increasing contraction frequency. 78

20

Symbiosis status 18 Aposymbiotic Symbiotic 16

14

12

10

8 Contraction Contraction Rate /(# hour)

6

4 0 1 2 3 4 5 6 7 8 9 10 11 12 1440 Time (hrs)

Figure 4.2: Variation in contraction frequency of symbiotic and aposymbiotic hydra in non-acclimated state (time 0hr, 18°C), acclimating state (time 1 to 12hrs, transferred to 30°C), and acclimated states (time 1440hrs, 30°C) with time.

Previously acclimated hydra (“Deacclimating” in Fig. 4.3) had higher thermal tolerance than acclimating hydra, especially acclimating symbiotic hydra

(F1,297=10.81, p=0.0011). Over the 21 days, persistence time at 38°C decreased for deacclimating hydra and increased for acclimating hydra (acclimation status effect:

F1,325=56.50, p<0.0001) but the change in persistence time did not depend on symbiosis status (F1,325=2.34, p=0.1267) or the interaction of acclimation and symbiosis status (F1,326=1.32, p=0.2512). After 60 days, hydra that acclimated 79 during this experimental period (“Acclimated” Fig. 4.3) had the greatest persistence time, hydra that deacclimated during this experimental period had intermediate persistence time (“Deacclimated” Fig. 4.3) and non-acclimated controls had the lowest persistence time (F2,107=80.73, p<0.0001). Symbiosis status affected persistence time only in interaction with acclimation status (main effect: F1,107=3.65, p=0.0586; interaction effect: F2,107=3.22, p<0.0439) with symbiotic controls having shorter persistence time than aposymbiotic controls.

Acclimated 100 90 Deacclimated 80

70 Deacclimating

60 Acclimation status 50 Acclimating

40 Persistence timePersistence (min) Controls Symbiosis status 30 Aposymbiotic Symbiotic

0 5 10 15 20 60

Time (days)

Figure 4.3: Thermal tolerance of hydra heat shocked at 38°C in states of 1) non-acclimated controls at 18°C (triangles). 2) acclimating at 30°C (circles). 3) acclimated after two months at 30°C (circles). 4) deacclimating that were transferred back to 18°C (squares). 5) deacclimated after two months back to 80

18°C (squares). Controls are the starting point for acclimating hydra. Acclimated hydra are the starting point for deacclimating hydra.

Without feeding for 20 days at 30°C, aposymbiotic hydra died more quickly than symbiotic hydra under sublethal heat stress (F1,16=19.31, p=0.0005, Fig. 4.4); on the contrary, all symbiotic and aposymbiotic hydra survived a similar period at

18°C (data not shown).

Figure 4.4: Survival of symbiotic and aposymbiotic starving hydra at 30°C after acclimation.

For the crossed hydra-algae reassociation experiment, survival analysis showed that survival time varied among hydra and algae treatments (chi-sq=77.5, 81 p<0.0001, 3df, Fig. 4.5) with LH.LA (both hydra and algae were not exposed to high temperature) survival differing from the other three treatments in pairwise comparisons (all p<0.0001) but the other three treatments did not differ from each other (0.38< p<0.96).

1.0

0.8

0.6

Survival 0.4

0.2 HH.HA LH.LA LH.HA HH.LA 0.0 0 12 24 36 48 60 72 84 96

Time (hours)

Figure 4.5: Thermal tolerance of reassociated algae and hydra: (1) Both hydra and algae experienced acclimation (HH.HA). (2) Both hydra and algae were not exposed to high temperature (LH.LA). (3) Only hydra experienced acclimation (HH.LA). (4) Only algae experienced acclimation (LH.HA).

4.4. Discussion

Adaptation and acclimation, the two major mechanisms that alleviate organisms’ stress in changing environments, have been proposed to be influenced 82 by both symbionts and hosts in holobionts [3, 117–119]. Our previous studies on green hydra thermal tolerance support that holobiont stress tolerance is controlled by both the host and the symbiont, and here we looked further into impacts of their plasticity [232]. We found once symbionts are acclimated, they will not limit hosts’ persistence under stress as previously suggested [8, 64, 249], although they could be detrimental initially during heat stress [232]; indeed, having symbionts turned out to be more advantageous during long-lasting sublethal heat stress when starvation co-occurs. We demonstrate that in addition to genetic variation in both the symbiont and the host [3], plasticity could also alter the holobiont phenotype and generate positive carryover effects over multiple asexual generations, adding another layer of complexity to hologenome theory, and imply that interactions may exist between alleles as well as epialleles of the host and the symbiont [287, 288].

Unlike results found in sea anemones (Exaiptasia sp.) and corals, we detected improved tolerance under heat stress at the holobiont level after acclimation [129,

267]. By measuring several distinct characteristics of symbiotic and aposymbiotic hydra, we found presence of algae had no negative impacts on hydra acclimation rate or thermal tolerance after acclimation (Fig. 4.2, Fig. 4.3). However, the more rapid decrease in contraction rate (Fig. 4.2) and lower persistence time of control symbiotic hydra (Fig. 4.3) indicate that symbiotic hydra are less thermally tolerant than aposymbiotic ones if they have no previous heat exposure, which is consistent with our past study [232]. The greater increase in persistence time of symbiotic hydra than aposymbiotic hydra gained through acclimation suggests the former has larger acclimation capacity. That the initially thermally sensitive algae became 83 thermally tolerant after acclimation could be a result of resource re-allocation. Algae allocate more energy to carbonate production than synthesizing heat-stable saturated fatty acids under normal conditions, which benefits hosts [289–291]. Once heat stress occurs, acclimation alone could mitigate the negative impacts of these symbionts on holobiont thermal tolerance, perhaps at the cost of supplying less carbonates to hosts [217, 292, 293].

The population response results also suggest being symbiotic does not impede hydra from acclimation, and can even become beneficial when stress is not transient.

While symbiotic hydra growth was greatly suppressed by elevated temperature initially, their growth rate became similar to that of aposymbiotic hydra after they were acclimated (Fig. 4.1). The growth of aposymbiotic hydra, on the other hand, remained relatively constant during and after acclimation. Having symbionts was more advantageous when starvation co-occurred with sublethal heat stress, as acclimated symbiotic hydra persisted for a longer time under these conditions than aposymbiotic ones (Fig. 4.4). This may reflect higher energy consumption by organisms at elevated temperature due to higher metabolic rates (indicated by higher contraction frequency in our experiment) [294, 295]. As the relative fitness of symbiotic and aposymbiotic hydra changed once acclimation takes place, we propose that: first, high and variable potentially lethal stress favors aposymbiotic organisms while symbiotic organisms suffer from discordant stress responses in the symbiont and the host; second, less intense stress favors symbiotic organisms as the acclimated symbiont provides supplementary functions to the host. 84

There is a concern that symbiotic species will be unable to withstand global climate change given high thermal sensitivity in the symbionts [8], but here we found symbionts may not limit holobiont persistence under prolonged sublethal heat stress as long as acclimation takes place. In our pilot experiments determining the appropriate acclimation temperature for hydra, we found algae but not hydra

(neither symbiotic or aposymbiotic ones) were able to survive at 35°C. Although stress responses have mainly been reported for symbionts under thermal stress, transcriptome analysis of transplanted corals showed that symbionts had significantly altered gene expression after one year of acclimation [49, 50, 133].

Thus a small portion of the remaining symbionts in hosts after heat stress may be able to achieve higher thermal tolerance after acclimation [191]. This is consistent with the idea that thermal sensitivity does not determine acclimation capacity [296].

Baker et al. [217] suggest symbionts may shift to parasitism in corals at elevated temperature, however, we show that short-term costs of hosting symbionts in acute environment changes appears to be offset once symbionts get acclimated. Because animals such as corals differ from hydra in within-polyp symbiont diversity, algal density stability, or transmission models, their responses to stress might not be the same as we have observed here [124, 189, 278, 284, 297–299]. Nevertheless, we showed that symbiotic organisms could ultimately persist in the most extreme conditions of which hosts alone are capable, but they may need more time to reach such maxima in tolerance given lower initial tolerance.

The heat sensitivity of algae did not reappear even after two months of deacclimation, as we found deacclimated symbiotic and aposymbiotic hydra had 85 similar thermal tolerances (Fig. 4.3). In fact, deacclimated hydra exhibited higher thermal tolerance compared with non-acclimated hydra for either symbiosis status, which further suggests the host also retained acquired thermal tolerance. The first rapidly and then steadily decreasing hydra thermal tolerance during deacclimation

(without acquired tolerance being lost) supports two different modes of heat acclimation: a short-term and a long-term one [260]. The improvement in heat resistance due to previous thermal exposure could be explained by the long-term mechanism that alters baseline stress responses [300]. However, whether the symbiont will eventually return to its tolerance baseline is unclear; it is possible that over longer timescales the symbiont loses acquired tolerance so the symbiotic hydra becomes less resistant to heat again.

We discovered heat resistance was conferred to holobiont offspring by acclimated hosts and algae independently (Fig. 4.5), exemplifying trans-generational plasticity exists in both parties over asexual generations. Although somatic mutations could occur during asexual reproduction of both the hydra and the algae in this study, we pooled all offspring together to evaluate their thermal tolerance and did not select for those with high heat resistance. Offspring of reassociated hydra, that received either heat exposed hosts or symbionts or both, had much greater thermal tolerance than the control group after three asexual generations at

18°C, so acclimation rather than adaptation is likely the main contributing factor to this increase in thermal tolerance [301]. This trans-generational plasticity has also been documented in other asexual species [273, 302–304]. For example, Verhoeven and van Gurp [275] discovered parental stress history would affect offspring’s 86 stress tolerance. Dewan et al. found temperature history on parents could affect phenotypes of asexually produced offspring [302]. Thus, for asexually reproducing holobionts, acclimation in either the host or the symbiont could benefit the holobiont as well as the offspring with future stress [305]. It implies that instead of experiencing heat stress directly, symbiotic species like corals could improve their thermal tolerance by receiving either hosts and/or larvae from heat-exposed colonies [300]. Nevertheless, the relative contribution of the host and the symbiont acclimation to holobiont stress tolerance improvement might not be equal as we found in our experiment, since acclimation responses differ among holobiont species [306].

The positive trans-generational effects we observed were probably caused by epigenetic modifications rather than maternal provisioning [242]. Because the reassociated hydra had been cultured at 18°C for three to four generations before their thermal tolerances were tested, any maternal provisioning effects should have already faded away [307]. Such positive trans-generational effects were also detected in potentially sexually reproducing corals as revealed by Putnam and Gates

[305, 308, 309], although they only tested for one offspring generation, and the larvae experienced preconditioning within parents. In addition, almost exclusively asexual reproduction of hydra and algae could promote epigenetic inherence [259,

283]. Since both the host and the symbiont only went through mitosis in this study, they could retain the same DNA methylation or histone modifications from their acclimated parents [259]. We suggest that maternal provisioning might influence immediate offspring fitness when the holobiont reproduces during stress, while 87 epigenetic modification could confer tolerance for multiple asexual generations even if the holobiont is stressed before but not during reproduction. This adds another layer of complexity on the phenotype of holobionts, which appears now to be controlled by the host’s and the symbiont’s genomes, their genome interactions, their epigenomes, and their epigenome interactions [3, 205, 287].

4.5. Conclusions

Our study experimentally verified that acclimation could occur in both the host and the symbiont, which benefits holobionts and their offspring under stress.

Although symbiotic hydra had lower thermal tolerance than aposymbiotic hydra before acclimation, they both had similar acclimation/deacclimation rates and thermal tolerance after acclimation, indicating symbiotic hydra have greater acclimation capacity. It was unexpected to find that the presence of symbionts increased hydra fitness in prolonged sublethal heat stress when paired with starvation. To the best of our knowledge, our study is the first to show positive trans-generational effects in both hosts and symbionts of asexually reproducing holobionts, which demonstrates that symbiont presence can promote host survival rather than constrain it under climate change. It should be kept in mind that we only manipulated algal symbionts in this study, and it could be possible that associated bacterial microbiota also contribute to hydra thermal tolerance and acclimation

[277, 310, 311]. Our work is based on a photosynthetic symbiosis model organism and thermal stress, yet the results may also be applicable to other kinds of abiotic stress and other symbiotic organisms. We propose symbionts provide hosts a dual 88 insurance under climate change when the initial change is not sudden and large as symbiont acclimation can significantly improve holobiont stress tolerance. It indicates a previously stressed population not only may more readily survive subsequent stress but also could disperse acclimated hosts and/or symbionts to other populations and buffer them from future stress. This might guide symbiotic ecosystem management (e.g., coral reefs) by implying that a natural area experiencing less intense stress may be a priority for preservation and may even facilitate preservation of others experiencing more intense perturbations.

89

Chapter 5

An agent-based model of adaptation of holobionts with different microbial symbiont transmission modes

ABSTRACT

The hologenome theory suggests that holobionts (host plus symbiont) with hosts that are only able to adapt slowly may be able to persist in deteriorating environmental conditions via rapid adaptation of their microbial symbionts. The effectiveness of such symbiont adaptation may vary depending on whether symbionts are passed directly to offspring (vertical transmission) or acquired from the environment (horizontal transmission). However, it has been suggested that holobionts with horizontal transmission cannot pass down their symbionts faithfully, preventing adaptation at the holobiont level because of host-symbiont disassociation between generations. Here we investigated whether holobionts with horizontal microbial symbiont transmission can adapt to increasing stress solely 90 through symbiont adaptation by using an agent-based model, and compared their adaptation to holobionts with vertical transmission. We found that holobionts with either transmission mode was able to adapt to increasing abiotic stress solely via symbiont adaptation. Moreover, those with horizontal transmission were more competitive than those with vertical transmission when hosts were able to selectively associate with the most suitable symbionts. However, those with horizontal transmission were less competitive than those with vertical transmission when symbiont establishment was random. Our results support the hologenome theory and demonstrate that holobionts with horizontal microbial symbiont transmission could adapt to increasing abiotic stress via their symbionts. We also showed that whether holobionts with horizontal or vertical symbiont transmission are favored in increasingly stressful conditions depends on the ability of hosts to recognize and foster stress tolerance conferring microbial symbionts.

5.1. Introduction

Research on symbiotic organisms has suggested that symbiont variation can drive holobiont (host plus symbiont) phenotypic diversification, and influence holobiont fitness [77]. In addition to host genetic changes, symbiont genetic changes can alter the holobiont phenotype and therefore impact holobiont adaptation [312].

Zilber-Rosenberg and Rosenberg proposed that rapid holobiont adaptation could take place even when host adaptation is slow, because symbiont adaptation and composition shift could be rapid [3]. A diversity of potential symbiont partners are 91 rich resources for holobionts, which might be critical for holobionts to adapt to increasing environmental stress such as global warming [93, 100].

Compared to hosts, symbionts have large population sizes and short generation times, and may adapt to ambient changes [10], yet it is unclear whether symbiont adaptation alone could drive holobiont adaptation. Most studies on holobionts focused on symbiont alteration rather than symbiont adaptation. Experiments on testing holobiont adaptation via symbiont adaptation and composition shifts would be hard because they require tracking of symbiont composition as well as holobiont phenotype over a long time. For example, researchers found cnidarians associating with different symbiont strains differ in their thermal tolerances [93, 232], and thermal tolerance of aphids can be controlled by manipulating their symbiotic bacteria [9, 73]. Such changes in holobiont phenotype are mainly based on existing symbiont diversity and can be observed within one generation. In contrast, whether symbiont adaptation could take place and affect holobiont phenotypes is less explored. Chakravarti et al. [127] pointed out that assisted evolution in symbionts can be applied to improve holobiont stress tolerance, and they were able to detect its impact on symbionts stress tolerance, but not on the holobiont stress tolerance.

Some field observations also suggest holobiont adaptation can be achieved through symbiont adaptation. Rodriguez et al. [22] discovered the stress tolerance conferred by a symbiotic fungal to host plants is habitat dependent. Another study on corals also revealed local adaptation existed in symbionts and leads to holobiont divergence in their thermal tolerance, in which the symbiotic algae collected from warmer reefs maintained higher resistance to heat compared to those from cooler 92 habitats even after multiple asexual generations [126]. Nevertheless, it is hard to monitor symbiont adaptation in the field over evolutionary time scales and track its impact on holobiont fitness. A simulation model would be appropriate to study holobiont adaptation via symbiont adaptation [313, 314], which is lacking in holobiont research.

To investigate whether holobionts can adapt to stressful environments depending on symbiont adaptation, it is critical to understand whether holobionts with either horizontal symbiont transmission (H) or vertical symbiont transmission

(V) are both able to pass their adaptive symbionts to offspring, and which type is better at coping with stress. Some researchers argue that passing down adaptive symbionts to offspring is unstable for holobionts conducting horizontal transmission, which makes the hologenome theory less plausible [141, 144]. They suggest the symbiont community could be shaped simply by opportunity and environmental filters, and the transmission fidelity is low [141, 144]. In addition, opposite opinions are given when discussing H’s and V’s adaptability. In vertical transmission, individuals inherit symbionts directly from parents, which is similar to classical gene inheritance, so adaptive symbionts from parents can be passed to offspring [97]. But there are drawbacks associated with this transmission mode; symbionts picked up by offspring might only be a subset of their parents’, so symbiont diversity could decrease dramatically through evolutionary time, and become maladaptive once the ambient conditions change [125, 156]. On the other hand, individuals conducting horizontal transmission pick up a variety of symbionts in the neighborhood, so they might respond to environmental changes faster by 93 associating with stress tolerant symbionts [98, 100]. However, the disassociation of symbionts and hosts between generations could lead to loss of adaptive symbionts and colonization of virulent symbionts [79, 315]. Most studies on microbe transmission and adaptation in the past investigated how different transmission modes affect virulence [97, 315–317], and explored the dynamics of virulence- transmission trade off instead of considering them as a selection unit [145, 318,

319], because their systems emphasize antagonistic rather than mutualistic relations between the host and the symbiont. Roughgarden [155] conducted a pioneering simulation of holobiont evolution, where she found both vertically transmitted and horizontally transmitted agents were able to evolve as a holobiont unit. But she used the number of microbes to determine holobiont fitness in her model, and did not study the interactions between the environment and holobionts.

Because of the complex interactions among the host, the symbiont, and the environment, combined with the different symbiont acquisition and retention mechanisms in Hs and Vs, it is hard to predict whether Hs and Vs are able to adapt to climate change, and which modes is more adaptive with increasing environmental stress. Holobionts with different transmission modes could differ in their abilities of novel symbiont acquisition and retention, which makes it hard to predict whether they are able to adapt to climate change, and which modes is more adaptive.

Here we use agent-based modeling to explore how holobionts with different transmission modes respond to increasing stress because such models are flexible and able to handle complex problems [320, 321]. Interestingly, such topic is more 94 well discussed in culture transmission, where researchers explore how knowledge information (e.g. language, knowledge, etc.) is transmitted between generations under different environment regimes [158]. In culture transmission, vertically inherited information (from parents) is more conservative than obliquely acquired

(from elder people excluding parents or ancestors) [322], which resembles vertically transmitted symbionts and horizontally transmitted symbionts. Some studies reveal oblique transmission is favored when the environment fluctuates, and is necessary in driving language evolution, while vertical transmission is preferred when the environment is stable [160, 161]. However, there could be fundamentally differences between cultural transmission and symbiont transmission. For example, there is no increasing stress such as global warming in culture transmission, so these studies do not provide insights into whether both transmission modes allow holobiont adaption to directional selection. In addition to transmission modes, we considered three other important factors that might impact holobiont persistence in changing climate: 1) the host’s ability to select for adaptive symbionts, 2) the rate at which stress increases, and 3) symbiont mutational variance.

We propose that the ability to select for adaptive symbionts is a key factor when studying holobiont adaptation, which is barely discussed, if at all, in previous research. Because the amount of resources and space an organism can provide to symbionts is limited, holobiont fitness is largely determined by the relative amount of adaptive symbionts within it [3, 323]. In contrast, there is little constraint on the amount of knowledge an individual can absorb, and he/she can choose the solution based on experience regardless how many different strategies he/she has learned. 95

So if symbiont acquisition is completely random, then Hs will have no advantages over Vs even if Hs have access to a larger symbiont pool from which they can choose partners, because the chances of getting adaptive and maladaptive symbionts are the same. In addition, drift might have larger impacts on Vs because the bottleneck effect is stronger in them due to the smaller effective symbiont population size [125].

On the contrary, if symbiont acquisition is not random but can be determined by the host, then Hs might be more likely to associate with adaptive symbionts than Vs, and drift impacts would be limited. Such symbiont selection by hosts is not uncommon and has be found in both Hs and Vs, for example, solitary wasps block transmission of nonnative symbionts to offspring, and sorghum selectively increases monoderm bacteria during drought to improve plant growth [324–326]. Thus we tested the impacts of drift on Hs and Vs adaptation by switching on and off the ability of holobionts to select for adaptive symbionts in our model.

Whether the holobionts are able to adapt to the changing environment via symbiont adaptation also depends on how fast the stress increases and how fast the symbionts adapt. Holobionts such as corals are assumed to rely on the symbiont rather than the host to adapt to rising sea temperature, because corals are living close to their thermal limits and the temperature is increasing quickly [327, 328].

Compared to the host, the symbionts are abundant with short generations, so they are more likely the key to holobiont adaptation [3, 122]. Because most symbionts

(e.g., bacteria, algae, fungi) reproduce asexually [127, 253], we consider mutation the major source of generating novel traits. Incorporation of mutation in our model makes the traits continuous and dynamic instead of static and discrete, and 96 generates intra-individual as well as inter-individual symbiont variation. Such variation is necessary in understanding the complex interactions between holobionts and the environment, and interactions among holobionts [329, 330].

Because population trait variance is sensitive to the magnitude of mutation (i.e., mutational variance) rather than mutation rate, we would like to know how mutational variance might affect distribution of symbiont traits and thus holobiont adaptation [331, 332]. Large mutational variance may have greater impacts on horizontally transmitted holobionts, because once an extreme symbiont strain arises, it can be transmitted throughout the population and may be hard to lose, while it will be constrained in certain lineages in vertically transmitted holobionts.

But large mutational variance could also produce lineages that have accumulated extreme symbionts in Vs when drift is strong. By varying the speed at which stress increases and mutational variance, combined with controlling drift, we explored how the selection-mutation-drift balance interacted with transmission modes.

Our goal was to present a model to test whether holobiont adaptation can be driven by associated symbiont changes, which involves symbiont mutation, acquisition, amplification and transmission. The agent-based model we used simulated changes at the environment level, the holobiont level, and the symbiont level, by varying four main parameters: transmission modes, the ability to select for adaptive symbionts, the rate of environmental change and the magnitude of symbiont mutation. It involves interactions between the environment and the holobionts, among the holobionts, and between the holobionts and the symbionts.

We believe our model could produce novel insights into holobiont adaptation. 97

5.2. Method

Our model was coded in python 3.0 with the agent-based modeling package

“Mesa”. Here we present a model description following the ODD (Overview, Design

Concepts, Details) protocol by giving a summary of the overall model structure and processes [320]. Detailed variables and functions definitions can be found in

Appendix A.

5.2.1. Purpose

The purpose of the model is to understand whether holobionts with vertical transmission or horizontal transmission could adapt to changing environments solely depending on their symbionts, and how drift and mutation would affect this process. The stress in this model is called “temperature”, which is a hypothetical stress without real world metric because it could also be called “salinity” or “UV intensity”. Similarly, each timestep represents one generation time, and has no corresponding real-world time length, because symbiotic species vary in their generation time. To minimize the model complexity, holobionts (the agents) in our model are autotrophic or heterotrophic with unlimited food and so do not need to compete for food, but they have to compete for space, which is common in ecosystems such as coral reefs or plants [122, 207]. Once a grid is occupied, it won’t be taken over by another agent unless the previous occupier is dead. 98

5.2.2. Scales and variables

The model simulates adaptation of Vs and Hs under different temperature regimes, which contains three hierarchical levels: 1) environment, 2) holobiont, 3) symbiont. The object of the environment level is temperature. The temperature is a standardized hypothetical parameter without units because we are not studying a specific species and a specific stress tolerance, but rather to verify if holobionts can evolve as a unit. Each time step represents one generation, and agents could die or give birth to one offspring. The objects of the second level are the two types of holobionts, Vs and Hs, which only differ in their transmission modes. To track the adaptability of Vs and Hs, agents reproduce asexually and do not switch their transmission modes across generations. This allowed us to better track symbiont compositions of the two agent types over time, and compare fitness of Vs and Hs.

The object of the third level, symbiont, is the only factor that determines holobionts’ fitness. Because our goal is to test whether both Vs and Hs can adapt via symbiont adaptation, and many holobionts’ stress tolerance is largely determined by symbiont stress tolerance [17, 243, 289, 328], we assume no host impacts on holobiont’s stress tolerance in this model. Each agent contains a fixed number of symbionts, and each symbiont is assigned a randomly generated gene value from which the host fitness will be calculated.

5.2.3. Scheduling

The model is executed in following steps: 99

(1). Model Initialization

Initially 2000 agents are generated and randomly distributed on a 50*50 size grid. In cases where both Hs and Vs are presented, each agent has 50% chance to be

H and 50% chance to be V. Each agent contains 50 symbionts, whose value is drawn from a normal distribution with mean equal to 0.5 and variance equal to mutational variance, truncated at two standard deviations [81, 332]. Initial temperature is set at 0.5 as well.

(2). Holobiont reproduction

At every time step, each agent has a chance to reproduce based on its fitness, which is the mean of the fitness score calculated for its symbionts, assuming all the symbionts within the host will affect the holobiont’s fitness. We used a symmetric beta distribution to calculate the fitness score [333], which reaches the maximum when the symbiont’s gene value matches current temperature (Fig. 5.1), and reaches a minimum when the mismatch is equal or greater than one, in other words, the current temperature could be either too hot or too cold for the symbiont. A random number is generated and compared to the holobiont’s fitness value to decide whether it will reproduce or not. Because conducting horizontal transmission might suppress holobiont reproduction, we introduce a cost parameter “l” that could reduce H’s reproduction probability [334].

100

Figure 5.1: Fitness curve. The fitness impact of each symbiont on the holobiont (fitness score) was calculated based the difference between the symbiont gene value (optimal temperature) and the current temperature. When the symbiont gene value matched the current temperature, the fitness score was set equal to 1 and it declined (beta function) as the gene value and temperature were more different. A positive mismatch value indicates the environment is colder than the optimal value for the symbiont and a negative value indicates the environment is colder than the optimal value.

(3). Symbiont transmission

In the default model, drift is introduced by enabling random symbiont transmission in Hs and Vs. V offspring sample symbionts from their parents randomly with replacement, so their symbionts could be a subset of those in their parents. H offspring sample symbionts from other agents around them once they are 101 produced and dispersed [97]. This transmission process is equivalent to picking up symbionts from a common pool constitute of symbionts released from agents [155].

In the optimal transmission model, drift is suppressed by enabling agents to pick the fittest symbionts from parents. As in cases where certain symbiont strains is dominant while the rest strains are marginal, here offspring choose the fittest strain from the parent (in V) or from neighboring agents (in H) to make up the symbiont population, and then randomly sample with replacement as in the default model to fill the rest of its symbiont population.

(4). Symbiont mutation

During holobiont reproduction, the symbiont may mutate by chance once it is acquired by the offspring. The mutation probability of each symbiont strain within the holobiont is set at 0.01 [335], given that symbiont generation time is shorter than holobiont generation time while the abundance is large [47, 336]. The mutated symbiont gene value is drawn from a normal distribution with mean centered on the original symbiont gene value, variance controlled by the mutational variance, truncated at two standard deviation from the mean [332]. Since we are interested in whether symbiont gene changes could drive holobiont adaptation, we assume heritability equals one and there is no environmental variance in our model.

(5). Holobiont dispersal

We use a stepwise function to control the position of a newborn holobiont.

Because the further away from the parent the less likely the offspring would occur 56102

[337], we set equally high probability within certain distance for the new holobiont to occupy the gird, and equally low probability beyond certain distance for the holobiont to occur.

(6). Selection

Agent fitness is calculated as described above, except for the decision whether the agent will die or not.

Detailed parameters are documented in appendix A, and each parameter combination was run for 4000 time steps and at least 20 iterations.

5.2.4. Design Concepts

Emergence: Population dynamics emerge from the reproduction, selection and competition of the two types of agents, embedded with symbiont drift and mutation. Adaptation is driven by interactions between each agent and the environment.

Sensing: Agents can perceive the temperature and know their transmission modes. In the optimal transmission model, agents are able to pick the fittest symbionts from their ancestors.

Interaction: Competition for space (empty cells) is modeled explicitly, based on a first come first serve rule. Reproduction and selection is simulated based on the beta functions [333] which depict the relation between fitness and temperature stress. 103

Stochasticity: At each process, the order of agents to execute the code is random. All behavioral parameters are generated based on empirical probability distributions. That means the reproduction, dispersal, and selection of agents are not deterministic, and symbiont values after mutation and inheritance in each agent are random. This introduces stochasticity on both symbiont and holobiont levels, and the final results are emergent from interactions between individuals and the environment.

Observation: For model analysis, population-level data were recorded, i.e., population size over time, relative ratio of the two agent types, mean holobiont optimal temperature, and time to extinction, etc. [338].

5.2.5. Simulation scenarios

(1) Test case

To test and validate the model, we constructed a basic model that both agent types have equal fixed reproduction and survival probability, so they are selectively neutral and their fitness does not depend on the environment. This could be viewed as two genotypes in a finite population, whose fixation should be random. According to Kimura & Ohta [339], when the initial relative ratio is 0.5, either H or V will fix at

50% chance, and if the initial relative ratio moves away from 0.5, one or the other will be more likely to fix within dramatically less time.

(2) Increasing temperature 104

The temperature was projected to increase at different speed infinitely, which means holobionts would only be able to persist by having novel mutations conferring thermal tolerance higher than that of existing symbionts. This model tested whether both Vs and Hs could keep up with increasing stress, and explored how symbiont mutational variance might affect this process given different increasing speed. For both random transmission model and optimal transmission model, Hs and Vs were simulated in coexistence and in separation.

5.2.6. Statistical analysis

We used paired Wilcoxon rank tests to compare the fitness between Hs and Vs across all the parameter combinations. We summed up the mean holobiont fitness at each time step of every iteration for a given parameter combination and performed a log transformation. This provides us information that includes both the fitness at each time step as well as the extinction time. We used the same test for Hs and Vs extinction time, correlation between holobiont gene values and the temperature, fitness score variance within holobionts, and fitness score variance between holobionts.

5.3. Results

5.3.1. Test case

We verified that when agents were selectively neutral in our basic model, the fixation was random at the 1:1 V to H initial ratio (Figure 5.2). 105

Figure 5.2: Fixation of holobiont transmission types under selectively neutral conditions and equal starting abundances of vertical and horizontal transmission types. Blue lines indicate the proportion of holobionts that have vertical transmission (V) and red lines indicate the proportion that have horizontal transmission (H). Each simulation is represented by a single blue line and single red line.

5.3.2. Increasing temperature

Regardless of whether transmission is random or optimal, both Vs and Hs were able to adapt to increasing temperature when the rate of temperature increase was low and mutation variance was high (Fig. 5.3).

106

a. b.

Gene Value/Temperature

Figure 5.3: Average symbiont gene values (optimal temperature value) of horizontal (H) and vertical (V) transmission mode holobionts in (a) the random transmission model with temperature rate of change per time step=0.001 and magnitude of mutation with standard deviation=0.1, (b) the optimal transmission model at step=0.001, mutation_sd=0.01.

Generally, holobionts persisted in high mutation variance and low

temperature rate of change conditions, but went extinct in low mutation variance

and high temperature rate of change conditions.

However, the transmission mode did affect the relative fitness of Hs and Vs

(Fig. 5.4). When transmission was completely random, Vs had higher fitness

(Wilcoxon test, p=0.0002) and longer time to extinction (Wilcoxon test, p=0.03)

compared to Hs for the same parameter combination, and their gene values also had

higher correlation with the temperature (Wilcoxon test, p=0.0017). When

transmission was optimal, Hs had higher fitness and longer time to extinction, as

well as higher correlation between gene values and temperature. 107

V H

a.

High fitness/ Slow extinction

Low fitness/ fast extinction

b.

Temperature change

Mutational magnitude (standard deviation)

Figure 5.4: The dependence of fitness and time to extinction of vertical (V) or horizontal (H) transmission holobionts in separate simulations on rate of temperature change (magnitude of change per time step) and mutation magnitude (standard deviation) with (a) the random transmission model, (b) optimal transmission model. Rapid temperature change (large change per time step) and small magnitude mutations (low mutation variance) are likely to lower holobiont fitness and lead to extinction. The scenarios with persistence are those with high average fitness and those with low fitness have rapid extinction. 108

When both agent types were present at the same time, we were able to compare the ratio of Hs and Vs (Fig. 5.5), which tells their relative competitiveness.

In both random transmission and optimal transmission models, Hs and Vs became extinct in large step/small mutation_sd regions, and coexisted in small step/small mutation_sd regions. In the rest of regions, Vs persisted in the random transmission model (Fig. 5.5a), and Hs persisted in the optimal transmission model (Fig. 5.5b).

a. b.

Temperature change

Mutational magnitude (standard deviation)

Figure 5.5: The dependence of average proportion of vertical transmission holobionts in simulations that include both vertical and horizontal transmission types on rate of temperature change (magnitude of change per time step) and mutation magnitude (standard deviation) with (a) random transmission, (b) optimal transmission. Brighter colors indicate conditions in which vertical transmission types reached fixation more quickly (and horizontal types became extinct) and darker colors indicate conditions in which vertical transmission types became extinct faster (and horizontal types reached fixation). 109

We also checked how virulence would affect the relative competitiveness of

Hs and Vs by reducing birthrate of Hs by 5% at mutation_sd=0.001 and step=0 in

both models. This resulted in Vs fixation in both models.

To explore how drift shapes symbiont distribution in the random transmission

model, we compared fitness variance between individuals and within individuals

(Fig. 5.6). We found Vs had larger variance among individuals (Wilcoxon test,

p<0.001), but less variance within individuals (Wilcoxon test, p<0.001). This means

symbiont distributions in each vertical holobiont were more homogenous than in

each horizontal holobiont, but vertical holobionts were more heterogeneous than

horizontal holobionts across the holobiont fitness landscape.

a. b.

Figure 5.6: Variance of symbiont fitness scores (a) within hosts (average of the variance of scores for symbionts within single hosts), (b) between hosts (the variance of the average scores of hosts). Each point is the variance in a single random transmission simulation with only vertical or horizontal transmission types after 1000 time steps. 110

5.4. Discussion

We present here an agent-based modeling that investigates the adaptability of vertically transmitted holobionts and horizontally transmitted holobionts to increasing stress through symbiont adaptation. By introducing intraspecific trait variation and enabling evolution, we were able to study how selection-mutation- drift affected the population dynamics of holobionts with different transmission modes. Our model clearly demonstrates that both Vs and Hs are able to adapt to increasing environmental stress via symbiont adaptation, and their relative competitiveness depends on whether symbiont transmission is random or not. As long as the stress does not increase at a speed that will drive the holobionts to extinction rapidly, symbiont adaptation alone could increase holobiont stress tolerance.

Our model suggests the answer to a long debated question: can horizontally transmitted species be considered as a selection unit even the symbiont disassociate with the host during the holobiont life history [141, 142]? We showed that although offspring do not necessary inherit symbionts from their parents directly, they could still acquire adapted symbionts. We found stress tolerance conferred by symbionts improved over time because the mean symbiont gene value increased along with increasing temperature. This was because holobionts whose symbiont values better matched the temperature were more likely to survive, and they passed their adapted symbionts to individuals in the next generation. In other words, because selection acts on the population level and drives the symbiont to confer higher 111 stress tolerance, individual holobionts sampled from a symbiont pool that was adapting, and thus became adapted. This process looks like a Lamarckian evolution because agents acquire stress tolerance by associating with stress-resistant symbionts horizontally [340], but it is actually Darwinian evolution on the whole symbiont population level because the symbiont gene frequency changes due to selection against maladapted holobionts. Osmanovic et al. [341] observed the similar pattern that long-term selection on increased host stress tolerance conferred by symbiotic bacteria, although they only employed vertical transmission during evolution in their simulation model. Acquiring adaptive traits via associating with horizontally transmitted symbionts is commonly observed in nature. In aphids, the secondary symbionts are considered to form a horizontal gene pool which facilitate aphid adaptation [342]. Redman et al. discovered fungal-free plants that received

Curvularia isolates (a type of fungal endophyte) from geothermal regions exhibited improved thermal tolerance, which also suggests stress tolerance could be acquired horizontally [20]. In addition, research on horizontally transmitted corals revealed surprisingly high fidelity in symbionts across generations, indicating unknown mechanisms that maintain symbiont stability in Hs [151]. Thus we propose that symbiont-mediated holobiont adaptation is plausible in both Hs and Vs.

Further comparison of Vs’ and Hs’ adaptability shows that whether holobionts are able to identify and partner with adapted symbionts or not is critical in determining their relative competitiveness. Researchers have given opposite opinions regarding which transmission mode is more advantageous because of following reasons: on one hand, Hs could exploit a broader range of symbionts, and 112 are likely to take up adaptive symbionts from the environment [48, 79], while Vs can only inherit parents’ symbionts, which would be maladaptive once the environment changes [343, 344]. On the other hand, adaptive symbionts could be passed down faithfully in vertical transmission, but get lost during horizontal transmission [100,

144]. Our results suggest both arguments are partially correct, but are case dependent because symbiont acquisition could be random or non-random, which has significant impacts on the outcome [345]. When transmission is random, Hs have no advantage even though the holobionts have access to a wider range of symbionts, because the chances of taking up adaptive and maladaptive symbionts are equal; but when they are able to recognize adaptive symbionts, they can pick up the fittest symbionts from a larger pool compared to the limited choices Vs have, which greatly improves their adaptability.

In random transmission situations, drift is stronger in Vs and can drive symbiont fixation in offspring lineages rapidly because they experience strong bottleneck effects given limited symbiont choices in parents [125, 339]; in contrast,

Hs offspring tend to maintain their parents’ living optimum according to the law of large numbers, because they sample from a larger symbiont pool [346]. Our simulation of Hs and Vs without selection has shown that symbiont similarity is higher within each V than within each H (i.e., within host symbiont variation is larger in H individuals than in V individuals), but lower among Vs than among Hs

(i.e., between-host symbiont variation is lower among Hs than among Vs). This is consistent with studies on corals, that symbionts among vertically transmitted corals are more differentiated, and symbionts among horizontally transmitted 113 corals are less differentiated [151, 157]. In psyllids, some hosts are found to harbor only clonal symbiont lineages due to their vertical transmission mode, and the low diversity within the holobiont suggests strong bottleneck effect [121]. Given enough time, vertically transmitted symbionts could become so specific in host lineages that they will have reduced genome sizes and cannot escape from hosts [347]. The strategies of Vs and Hs are thus different when facing environmental changes. Vs produce lineages that are distinctive in their stress tolerance, while Hs produce individuals that try to persist in all conditions. Because maladaptive lineages that are dominated by maladaptive symbionts can be removed quickly from V populations, while maladaptive symbionts can persist in H populations through coexisting with adaptive symbionts, Vs are able to respond to environmental changes faster than Hs, and thus adapt faster when symbiont transmission is random.

Moreover, extremely maladaptive symbionts could be transmitted across the whole H population, while they are constrained in certain lineages in the V population, because of the larger potential host population available for each symbiont in Hs [79]. From this perspective, having maladaptive symbionts is similar to having parasites, which may provide additional explanation for why higher virulence is often associated with horizontal transmission [317, 319]. Because maladaptive symbionts can be acquired by holobionts other than direct descendants, they could persist in Hs just like infectious diseases, and decrease Hs’ overall fitness.

In contrast, V individuals have a smaller sampling pool, which is determined by their parent, and offspring that are quickly dominated by maladaptive strains due to drift 114 can be selected against [339], so overall fitness improves faster under selection. In short, once maladaptive symbionts arise in Hs, they can be taken up and passed down in the population and are hard to eliminate, but in Vs they can be trapped within lineages and be selected against, causing lower overall fitness in Hs than Vs.

In an extreme scenario when the stress increasing speed is zero (i.e., under a constant environment), although both populations could persist for the whole simulation, Vs had slightly higher fitness especially when the mutational variance is large, where maladaptive symbionts are likely to arise and spread in Hs. Given enough time, Vs should be able to outcompete Hs (Fig. 5a). This resembles results of a study on virulence in pathogens, where vertical transmitted pathogens were selected for to prevent host from acquiring more virulent pathogens horizontally

[319]. Our results of random transmission model are also consistent with previous studies on culture transmissions, which suggest Vs are favored under stable conditions [159, 161], and support the hypothesis that vertically transmitted holobionts such as corals may be more robust to climate change [348].

When we incorporated optimal symbiont uptake into the model, surprisingly we had the opposite result that Hs were better at following the environmental changes. Such “recognition” of suitable symbionts is not well explored in previous studies [155, 160], yet their impacts on the outcome is significant. We think such optimal transmission is equivalent to the positive transgenerational feedback proposed by Xue and Leibler [162]. They suggested phenotypes of parents are reinforced in offspring, so organisms could adapt to varying environments. The positive transgenerational effect has been observed in some empirical studies, 115 which is probably caused by epigenetic processes [301, 349]. Our previous experiment on green hydra also proved the positive transgenerational effects could occur in holobionts simply via transmission of acclimated symbionts. The model presented here is slightly different from that described in Xue and Leibler’s and examples mentioned above, because we are focusing on symbiont evolution rather than host evolution, and the phenotypes are distributed continuously rather than discretely. Nevertheless, the acquired symbionts were derived from those hosted by holobionts in the previous generations, which were more likely to be decedents of adaptive symbionts than maladaptive symbionts in their ancestors. Because the temperature in our model changes in one direction, the fittest new mutants are more likely to arise from the dominant, fittest strain in parent, instead of from a less fit strain. Such transmission of stress-tolerant symbionts has been observed in corals, which suggests holobiont adaptation can be achieved through transmission of adaptive symbionts [152, 350]. Having access to symbionts released by holobionts other than their direct ancestors, Hs were able to pick the fittest strain that arose in the population in contrast to Vs. The ability to pick up the fittest symbionts, which is similar to copy from the most successful individuals in social learning, increases Hs fitness and allows them to adapt faster than Vs [351].

There are two potential mechanisms that hosts are able employ to take in the fittest symbionts: the first is via partner choice; the second is via passive up take.

Mounting evidence of partner choice are found in holobionts [352], for example, plants allocate more resources to more beneficial partners and punish more parasitic partners [326, 353], so they could avoid cheaters and maximize their 116 fitness during symbiosis. Bobtail squid are able to prevent colonization of deficient luminescent bacteria Vibrio fischeri in their light organ, which could be another case of partner choice [354]. Massive coral bleaching could also be a case of hosts ejecting maladaptive symbionts and establishing symbiosis with adaptive ones

[328]. Corals are able to shift their symbiotic algae from sensitive strains to stress tolerant strains, so they can become more robust to stress such as high temperature and UV [5, 48, 82, 92]. Thus even coral hosts have long generation time and may not be able to adapt to warming temperature fast enough, corals still have a chance to withstand climate change [3]. An interesting example of wasps also shows that parents rather than the offspring may be able to choose symbionts to be transmitted

[325], as such transmission could be controlled on a cellular level [150]. In addition, even if the host is not able to identify the appropriate partner, they could form symbiosis with symbionts that pass an environment filter. Because symbionts vary in their stress tolerance, a strain that exhibits the highest fitness under certain conditions could outcompete other strains, and then form symbiosis with hosts [91].

Hosts taking up these adaptive symbionts would acquire appropriate stress tolerance towards their ambient environment [126]. In insects, thermal stress could easily wipe out their thermal sensitive symbiotic bacteria, which could reduce their fecundity [8, 9]. However, there are strains that could withstand such stress, and introducing these strains into hosts could restore hosts’ reproduction and enhance their thermal tolerance [9, 71]. Symbionts that diverge in their niches are able to confer different traits and form association with hosts in distinctive conditions, which facilitate holobiont diversification [73, 101, 342]. These examples provide 117 solid evidence of optimal transmission of symbionts and suggest we should take optimal symbiont transmission into consideration when studying holobiont persistence in stress.

Regardless of the transmission modes and the ability to associate with optimal symbionts or not, holobionts were more likely to persist under stress when their mutational variance was large and stress increasing speed was low. It is not surprising that holobionts were able to persist longer at low temperature increasing speed, because other studies also suggest rapid increase in stress could quickly wipe out the organisms [331, 355]. However, Ayllón et al. [332] did not detect significant impact of mutational variance on adaptation, and they suggested it was because their simulation ran for a limited number of generations (barely over 100 generations). In contrast, our model lasted for 4000 generations. Moreover, whether holobionts went to extinct or not seemed to be confined by non-linear combinations of the mutational variance and temperature increasing speed, which requires larger increase in mutational variance at high temperature increasing speed than that at low temperature increasing speed (Fig. 5.4).

Our models are the first exploring holobiont adaptation to stress, and demonstrate that both Hs and Vs are able to adapt, even when symbionts and hosts are disassociated between generations in Hs. Yet the model could be further expanded by incorporating more factors that might affect the dynamics between Hs and Vs in different scenarios. For instance, conducting horizontal transmission could be virulent, which might reduce Hs’ overall fitness and their competitiveness [319]. 118

In jellyfish, horizontal transmission favored symbionts shift to being parasitic, which reduced the growth rate of jellyfish up to 50% [315]. We have applied this only to scenarios when there is no change in temperature, and found a 5% decrease in Hs’ growth rate could greatly reduce their relative frequency in both random transmission and optimal transmission. This implies that under constant environments, vertical transmission with less virulent symbionts would be favored

[97, 159, 161, 334]. But if we adjust the virulence parameter in temperature increasing scenarios, we will be able to explore how virulence could alter holobiont population dynamics. Another factor worth exploring could be the tradeoff between thermal tolerance and fitness cost. Studies on corals reveal that symbionts that confer higher thermal tolerance may slow down the growth of corals [203, 356].

Imposing such constraints may enhance the authenticity of the model because holobionts will not be able to increase their thermal tolerance infinitely. We can also investigate into how Hs and Vs react in other scenarios such as random environments or fluctuating environments. We hope our models could provide a basic framework for holobiont evolution and shed light on symbionts’ roles from both ecological and evolutionary perspectives.

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Appendix A

Chapter 5 Model parameters

Parameters Description Initial Global Setting grid size: width, height define the whole map size initial population size: N define the initial population size Environment level temperature: env_press record current temperature temperature change speed: step define how fast the temperature change each step Holobiont level Agent type: gene_type agent is either H or V, denoted as 0 or 1 symbiont number in each host: number of symbionts strains per gen_info_size host lifetime :Life_time define how long one agent can survive dispersal distance: IMPECT_R define the dispersal distance and H’s symbiont sampling range virulence:l a coefficient that reduces H’s birth rate Symbiont level gene value: gen_info optimal growth condition of holobiont due to symbiont mutational variance: mutation_sd magnitude of mutation