The most primitive metazoan animals, the placozoans, show high sensitivity to increasing ocean temperatures and acidities

Abstract The increase in atmospheric carbon dioxide (CO2) leads to rising temperatures and acidification in the oceans, which directly or indirectly affects all marine organisms, from bacteria to animals. We here ask whether the simplest—and possibly also the oldest—metazoan animals, the placozoans, are particularly sensitive to ocean warming and acidification. Placozoans are found in all warm and temperate oceans and are soft‐bodied, microscopic invertebrates lacking any calcified structures, organs, or symmetry. We here show that placozoans respond highly sensitive to temperature and acidity stress. The data reveal differential responses in different placozoan lineages and encourage efforts to develop placozoans as a potential biomarker system.

sensitive (e.g., Foster, 1971;Pechenik, 1989). Rising water temperatures can also drive behavioral changes at the community level. To name just two out of many examples: The timing of spawning in the marine bivalve, Macoma balthica, is temperature dependent and so is the strength with which the sea star Pisaster ochraceus interacts with its principal prey (habitat forming mussels; Sanford, 1999). For the placozoans, which are found in most temperate and warm marine waters, nothing has been known yet about their sensitivity to temperature stress.
Overall, the literature on documented effects of rising temperature and acidity on marine invertebrates is limited, but nonetheless covers a broad spectrum of levels of observation and sensitive taxa (Table 1). The shown summary table documents how fragmentary our current is. Evolutionary constraints are part of every organism, but the limitations for adaptation to environmental change are hard to foresee. Moreover, little is known about combined effects of ocean warming and acidification on the development of marine invertebrates.
Combined effects of such stressors are not necessarily cumulative, because both additive and antagonistic (stress decreasing if combined) effects are known (Byrne & Przeslawski, 2013;Folt et al., 1999). Such effects have been studied in corals, mollusks, echinoderms, and crustaceans, across different ontogenetic stages. Additive negative effects on fertilization or growth rate, respectively, have for example been reported from the coral, Acropora tenuis, (Albright & Mason, 2013) and the oyster, Crassostrea gigas (Parker, Ross, & O'Connor, 2010).
Antagonistic effects have been found for example in the sea urchins Heliocidaris tuberculata (Byrne et al., 2010) and Sterechinus neumayeri Ericson et al., 2011), where warming partially compensated for the negative effect of acidification on larval growth.
In this study, we investigate the effects of temperature and acidity stress on placozoan reproduction and report strong and differential effects for both factors on the population growth rate (PGR) in different lineages (species) of placozoans. The observed differential sensitivity of different placozoan species or lineages suggests that placozoans might be promising organisms for developing a new generation of biomonitoring systems.

| Study organism
The phylum Placozoa holds a key position in the metazoan Tree of Life, close to the last common metazoan ancestor. Placozoans represent the simplest (not secondarily reduced) metazoan bauplan and have become an emerging model organism for understanding early metazoan evolution (Eitel et al., 2013;Schierwater, de Jong, & DeSalle, 2009;Schierwater, Eitel, et al., 2009;Schierwater et al., 2016;Signorovitch, Dellaporta, & Buss, 2006). These tiny invertebrates are common in warm tropical and subtropical as well as in some temperate marine waters in different depths up to 20 m. Their preferred habitats are calm water areas with hard substrates like mangrove tree roots, rocks, corals, and other hard substrates in the eulittoral and littoral zone. Placozoans have occasionally also been found on sandy surfaces or in areas with high wave activity.
At present, the phylum Placozoa is the only monotypic phylum in the animal kingdom, with the only formally described species Trichoplax adhaerens (Schulze, 1883(Schulze, , 1891. Placozoans offer unique possibilities for experimental ecophysiological studies because of their small size, simple morphology, and fast vegetative reproduction Eitel et al., 2011Eitel et al., , 2013Schierwater, 2005).
Vegetative reproduction through binary fission or budding is the usual mode of reproduction in the laboratory and in the field. In contrast, bisexual reproduction is rarely seen in the laboratory, but most likely present in all placozoans (Eitel et al., 2011;Signorovitch, Buss, & Dellaporta, 2007). The details of sexual reproduction and embryonic development in placozoans remain widely unknown, because all efforts to complete the sexual life cycle in the laboratory have been unsuccessful, because embryonic development has never gone beyond the 128 cell stage (Eitel et al., 2011). As the overall effects of physiological stress are best seen in the performance of vegetative reproduction by binary fission, we used overall PGR as the dependent and easily quantifiable variable for the subsequent experiments.

| Experimental setup for temperature experiments
All animal lineages used in the experiments have been cultured in our Institute of Animal Ecology and Cell Biology of the TiHo, Hannover (Germany), for several years: 1. H1-Trichoplax adhaerens (cosmopolitic), our so-called Grell lineage found by Karl Gottlieb Grell in an algal sample from the Red T A B L E 1 Summary of temperature and ocean acidification effects on marine biota in current literature

Major group Studied organism Effects of temperature Effects of pH Reference
Macroalgae Amphiroa fragillisima Decrease in calcification Langdon et al. (2003) Chondria dasyphylla

Echinometra mathaei
Early development  Affects growth Shirayama and Thornton (2005) Male spawning ability  Hemicentrotus pulcherrimus Early development  Affects growth Shirayama and Thornton (2005) Pisaster ochraceus Affects keystone predation Sanford (1999) Psammechinus miliaris Hypercapnia and mortality Miles et al. (2007) Strongylocentrotus franciscanus Experimental groups tested at 25 and 29°C were placed in separate aquaria (in the same room), filled with ASW (artificial seawater), and heated to the desired temperature by two heaters (ProTemp S200, accuracy: ±0.5°C). To keep the water temperature evenly distributed within aquaria, a water pump was installed to circulate the water (Figure 1).
At the start of the experiment, 360 individuals per lineage were randomly assigned to nine experimental groups (Table S1). Testing three lineages of placozoans, each for three different temperatures, we performed eight replicates with each five specimens as a starting point. After an acclimation period of 2 days (the chosen placozoan species adapt very quickly to new culture conditions), and in order to measure the PGR over the 3 weeks experimental period, the total number of individuals per plate was counted every 3 days (nine censuses).

| Experimental setup for pH experiments
We used the same lineages as described above. The aquarium was setup with a CO 2 reactor (JBL ProFlora), a pH meter, and an aeration system for the seawater carbon dioxide (CO 2 ) and the manipulation of the pH (for further details, see also Riebesell et al., 2000 and Figure 2).
At the start of the experiment, 80 specimens per lineage were randomly assigned to six experimental groups (Table S2). Food was provided ad libitum by placing one slide covered with algae inside the Petri dish. After an acclimation period of 2 days, the placozoans were left in one of two 160-L aquaria, one with a constant pH of 7.6, and the other with a pH of 8.0 (control; normal pH conditions in the laboratory cultures). In order to measure the PGR during the experimental period (12 days), the total number of individuals per plate was counted every 2 days (five censuses).

| Statistical analysis
The Kolmogorov-Smirnov one-sample test was used to test for normality distribution. As none of the data sets showed normal distribu-

F I G U R E 1
The experimental setup for the temperature experiment. 1-Aquarium filled with artificial seawater, 2-heater, 3-glass bowls turned over, 4-covered Petri dishes with the experimental animals placed on the glass bowls, 5-surface line of artificial seawater

| RESULTS
Both factors, temperature and pH, affected the PGR of different placozoan lineages significantly.

| The effect of temperature
The three lineages H1 gre, H2 ros , and H2 pan responded in sharply different ways to changes in water temperature: 1. The cosmopolitic H1 gre : One-way ANOVA revealed highly significant differences in the PGR for the three different temperatures (F 2, 27 = 14.89, df = 2, p < .001). Post hoc tests revealed highly significant differences in the PGR between 25 and 29°C (p < .001) and also between 21 and 25°C (p = .013). Between 21 and 29°C, no significant difference was observed (p > .05); at both temperatures, the PGR was low compared to the "optimal" temperature of 25°C (Figure 3a).

The cold-water H2 ros :
Also here, the effect of temperature on the PGR was significant (F 2, 27 = 8.04, df = 2, p = .002; one-way ANOVA). Post hoc tests revealed significant differences in the PGR between 21 and 29°C (p = .002) and also between 21 and 25°C (p = .033), while between 25 and 29°C, no significant difference was observed (p > .05). At both higher temperatures, the PGR of the cold H2 ros was low suggesting the lower temperature of 21°C to be preferred (Figure 3b).

The warm-water H2 pan :
The H2 pan clone behaved similar to the H1 gre clone, showing significant changes in PGR when moving away from the "optimal" temperature of 25°C (F 2, 27 = 6.08, df = 2, p = .007; one-way ANOVA). The harmful effect of higher temperature even on the warm-water population seems particularly notable (Figure 3c).
Profound effects of slight changes in pH value were found for the lineages H1 gre and H2 ros . After about 5 days into the experiment, the PGR in the acidified water slowed down significantly compared to the control (pH 8.0) cultures, with the effect becoming more and more substantial over time (Figure 4a-c and Table 2). The Panama lineage showed an unusual slow PGR under the given conditions (room temperature-21°C) already at "normal" pH conditions. As we do not know the reasons for the unusual slow reproductive activity, we excluded these data from further analyses. The observation that under more acid conditions, the PGR was higher than under pH 8.0 conditions maybe an artifact or may indeed be a lineage-specific adaptive response, but at this point, any further conclusions would be premature. . Mean global temperatures will continue to rise even if greenhouse gas emissions are stabilized at present levels (IPCC, 2001(IPCC, , 2013. Some of the most affected ecosystems are the oceans, which show rising temperature and acidity. Sensitive organisms, which respond to such changes early and are restrained from quick adaptations by evolutionary constraints, might be useful biomarkers for biomonitoring studies (e.g., Dallas & Jha, 2015;Moschino, Del Negro, & De Vittor, 2016;Natalotto et al., 2015) .

| DISCUSSION
Our experiments revealed strong and differential effects of both, temperature and pH, on the PGR of placozoans, with temperature showing the strongest effects. Interestingly, but not surprisingly, the lineage which had been found in relatively cold Atlantic waters (H2 ros ) showed a thermal preference for the low temperature setting, whereas higher temperatures significantly reduced the PGR. The other two lineages performed best at 25°C, which has been regarded as the "normal" temperature for placozoans (Schierwater, 2005). Both, T. adhaerens (species H1 gre , which has been collected from the Red Sea) and H2 pan (collected from Panama), only performed well at 25°C. Interestingly, for clones adapted to tropical waters, both species almost cease propagation at the high temperature of 29°C. As all clones sharply reduce propagation rates at the highest temperature, we assume harmful effects of such high temperatures for placozoans in general.
Placozoans behave like most marine species, which show thermal preferences for a well-defined temperature range (IPCC, 2007;Nakano, 2014). In many locations, ocean temperatures have either increased (Bethoux, Gentili, & Tailliez, 1998;Freeland, 1990;IPCC, 1996, Ji et al., 2007Scranton et al., 1987) or decreased in short time (IPCC, 1996, Ji et al., 2007Read & Gould, 1992), and demographic effects on many marine species, including placozoans, must have occurred recently. According to Hiscock et al. (2004), the ocean temperature will continue to show significant short-term variations, with maximum ocean-surface temperatures close to 28°C (with a trend toward even higher temperatures). As the natural habitat of placozoans is mainly surface waters, we must predict ongoing demographic changes and differential effects on placozoan communities.
Such differential effects mark placozoans as potential biomarkers for monitoring studies on the effects of ocean warming.
The sharp decline in propagation rate observed in T. adhaerens (H1 gre ) and H2 ros mirrors a quite sensitive response to increasing water acidity. This sensitivity is also highlighted by quite extreme changes in morphology toward the end of the experiments ( Figure 5). conclusive, the relative increase in PGR toward the end of the experiment as well as the differences between the other two clones suggests that different placozoan lineages differ in their sensitivity and response to change in water acidity. These observations not only highlight the sensitivity of placozoans to water acidity but also point to the potential of combining different sympatric placozoan species into a multiple-

| Final conclusions
Placozoans, the most simple organized and possibly also the oldest metazoan animals (cf. Schierwater, de Jong, et al., 2009;Schierwater, Eitel, et al., 2009), are highly sensitive to temperature and acidity stress and thus might be explored as potential biosensors. They offer the unique advantage of showing differential response patterns in different but sympatrically occurring placozoan species. The potential of a multiple "cryptic" species monitoring system has not been explored yet, but in practice should be based upon high-throughput genetic assays of community diversity and stress gene expression.
Furthermore, the quantified differences in niche parameters must also be relevant for species descriptions following the taxonomic circle approach in a large group of cryptic placozoan species.

CONFLICT OF INTEREST
None declared.

F I G U R E 5 Changes in morphology of
Trichoplax adhaerens under acidity stress.