Oxygen Limits of Sponge Respiration.

Overall, the patterns of oxygen uptake observed during the initial drawdown to anoxia followed Michaelis–Menten kinetics, with a V_max of 2.42% air saturation/h⋅g_{dry weight}, or 7.93 μM O₂/h⋅g_{dry weight}, and a K_m of 4.86% of air saturation, or 15.9 μM O₂, at the water salinity and temperature of our experiments (Fig. 3). This K_m is less than half of the 10% PAL serving as the estimated minimum level of atmospheric oxygen thought to have permitted the oxygenation of the deep oceans around 580 Ma (2), as well as the level estimated to permit the existence of millimeter-width, diffusion-limited animals (15).

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Fig. 3.

Michaelis–Menton kinetics: the maximum respiration rate, V_max, was 2.42% air saturation/h⋅g_{dry weight} or 7.93 μM O₂/h⋅g_{dry weight}, and K_m, the oxygen level where the respiration rate is half of V_max, was 4.86% of air saturation or 15.9 μM O₂.

Characteristic examples of the respiration and feeding rates of H. panicea at the different oxygen levels maintained during active oxygen regulation are shown in Fig. 4. Some oxygen respiration was apparent during the sponge-free controls at the end of the experiment, amounting to 25% of the rates measured in the presence of living sponges (Fig. 4B). Some clearance of Rhodomonas sp. was also observed during postexperiment negative controls, at 14% of the rates observed while the sponges were active (Fig. 4C). Therefore, the majority of the oxygen uptake and Rhodomonas consumption observed during the experiments can be attributed to the sponges, with the background rates likely attributed to free-living microbes in our experimental system. Overall, the sponges were able to both respire and feed to oxygen levels cycling between 0.5% and 4% PAL. These were the lowest levels we could maintain without driving the sponges into anoxia during part of the oxygen control cycle.

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Fig. 4.

Respiration and feeding under low oxygen. (A) Respiration curve showing oxygen regulation around a set point of 2.5% air saturation (indicated by the horizontal dashed line). The respiration rates are equal to the slopes of each downward curve (indicated by the diagonal dashed lines). (B) Oxygen consumption rates of a single sponge under different ambient oxygen levels. The areas marked with NC represent measurements taking during the sponge-free negative control. The horizontal line marks the maximum respiration rate observed under the negative control. (C) Clearance rates of Rhodomonas sp. cells under different oxygen concentrations. The area to right of the vertical dashed line designates the sponge-free controls. The horizontal dashed line marks the maximum clearance rate achieved during the negative controls.

While the sponges continued to feed and respire above background rates under the lowest oxygen range we could control, in many cases, the sponge showed visible signs of cell death (or “necrosis,” noted by dark patches of dead tissue) by the end of the experiment. This motivated us to assess whether this degradation of sponge health was (i) an experimental artifact due to the accumulation of sponge waste during the experiment, (ii) a problem with long-term sponge survival at low oxygen, or (iii) a general problem with keeping sponges in captivity within this particular setup for prolonged periods. We addressed this by constructing our second setup.

With our second experimental setup (Fig. S2), a relatively small sponge with numerous branching structures completely retained its healthy appearance under incubation at 3–4% PAL oxygen for a full 24 d, after which the experiment was terminated. Indeed, this individual even displayed signs of growth, evidenced by the elongation of several of its smaller protrusions (Fig. S3) and by the growth of new biomass on the walls of the incubation container (Fig. S4). Given the complete absence of necrosis in the sponges maintained at both low and high oxygen levels during this run, the necrosis observed during experiment 1 was likely due to the infrequent exchange of fresh seawater and not necessarily the low oxygen levels.

However, during another run of experiment 2, the sponge kept at 3–4% PAL developed necrosis after its water became inadvertently anoxic for a period of 4 h on the ninth day of incubation (Figs. S3 and S5). This condition persisted for the next 5 d of the experiment under 3–4% PAL oxygen. The necrosis, however, disappeared when the sponge was reintroduced to fully oxygenated water (Fig. S5). This suggests that although 3–4% PAL oxygen may not compromise H. panicea’s growth or maintenance of healthy tissue, anoxia still does.

Our results suggest that H. panicea is sensitive to both anoxia and prolonged exposure to relatively stagnant seawater. The sensitivity of sponges to water stagnation is not too surprising because sponges are notoriously difficult to maintain in aquaria, possibly due to the accumulation of metabolic products that interfere with their metabolism. The sensitivity to anoxia is also not surprising because H. panicea is an aerobic organism. Moreover, like many other sponges, H. panicea hosts a diversity of symbiotic microbes, and in the case of our sponges, functional gene and 16S rRNA analyses reveal a significant population of ammonium-oxidizing Archaea and betaproteobacteria, as well as other aerobic organisms (Fig. S6 and Table S1). Internal anoxia, however, does appear to be a common feature of sponge physiology (34, 35). This suggests that sponges, in general, are adapted to endure internal low-oxygen conditions.

H. panicea is classified as a low-microbial abundance (LMA) sponge (36, 37). LMA sponges are known to have shorter and wider water canals (38), as well as larger choanoflagellate chambers (39), than high-microbial abundance (HMA) sponges. Presumably, this allows LMA sponges to better maintain oxic conditions within their interiors because the surface area to volume ratio of their water canals is relatively low. Because of this, as water pumps through the water canal system, less oxygen diffuses out of the canals. In contrast, narrower water canals, as found in HMA sponges, have higher surface area to volume ratios, allowing relatively more oxygen to diffuse out of the canals during seawater pumping. It would appear, then, that LMA sponges, such as H. panicea, may be better adapted to low-oxygen conditions than HMA sponges. Although we did not test for this explicitly, our observations are also consistent with the idea that smaller sponges have an increased tolerance to low-oxygen conditions relative to larger sponges. This idea makes sense as smaller sponges have shorter water canals, which should result in less oxygen drawdown as water is pumped through the sponge. Taken together, it seems plausible that even smaller sponges, especially those with a low microbial abundance, could metabolize and grow under even lower oxygen concentrations than the 0.5–4% PAL range explored in our experiments.

Sponge Evolution and Environmental Oxygen Levels.

Recent molecular clock estimates place the last common ancestor of all sponges at nearly 800 Ma (12), whereas putative sponge-like body fossils are found in rocks dated between 660 and 635 Ma (10) in the Cryogenian Period (850–635 Ma). Elevated levels of possible demosponge biomarkers are also reported from rocks of similar age (9), but there is ambiguity as to whether these biomarkers truly originate from sponges (40). Unequivocal sponge body fossils are only first described in the lower Cambrian Period (41, 42). Nevertheless, if the body fossil and/or biomarker evidence are substantiated as true sponge fossils, they would reinforce the molecular clock divergence estimates and suggest that sponges evolved sometime during the Cryogenian Period. Such an early evolution for sponges would be well before the general oxygenation of the deep oceans in the Ediacaran Period.

Although levels of atmospheric oxygen may have reached 10% PAL by 580 Ma, the geochemical record does not preclude oxygen levels of 0.5–4% PAL during the Cryogenian Period (43, 44). Indeed, oxygen levels this high may have been reached as early as the Great Oxidation Event some 2.3–2.4 billion years ago (43, 45) and may have persisted at these levels through much of the intervening time (43, 44), with some likely swings to lower levels, however, around 1.8 billion years ago (46). Oxygen levels supportive of sponges may have been met even earlier, at least locally, in Archean oxygen oases (47).

Therefore, it is possible that the oxygen content of the atmosphere was completely permissive to the origin and early evolution of sponge-grade metazoans well before their evolutionary first appearance. This would mean, in turn, that the origin of animal life did not require a concurrent oxygenation event. Indeed, biological developments, such as the evolution of sophisticated gene regulatory networks, may have controlled the timing of animal origins more so than environmental parameters (12, 19, 48). Overall, animal-grade multicellularity likely required the accumulation and integration of specific traits, including cell adhesion molecules necessary for multicellular body plans, signaling proteins for cell–cell communication, and transcription factors essential for coordinated development (49). The accumulation of these traits and not the enhanced availability of oxygen may have controlled the onset of animal evolution.

Is there, then, any causal relationship between the geochemical record of increased ocean oxygenation during the late Neoproterozoic Era and animal evolution? It is argued that before the advent of suspension-feeding metazoans and large export-prone eukaryotic phytoplankton, the relatively slow sinking of organic detritus in the oceans resulted in higher water turbidity, with the decomposition of this slowly settling organic matter enhancing water column anoxia at shallow depths (48, 50⇓–52). The evolution of large planktonic eukaryotes and the evolution of animals themselves may have changed this dynamic by producing large, rapidly settling particles, such as the tests of large plankton and the fecal pellets of pelagic animals. This rapidly settling organic debris would have promoted the decomposition of surface-derived organic matter at greater depths, producing less oxygen demand in the upper regions of the ocean and allowing, in turn, for the greater oxygenation of these waters (51, 52). In this way, the geochemical proxies used to track ancient marine redox conditions may be revealing a biologically induced ventilation of the Neoproterozoic oceans, as opposed to an oxygenation of the atmosphere (52). Under this scenario, the origin of animal life was not a response to atmospheric oxygenation but part of the ecological reworking of marine biogeochemical cycles.