Explanation of Boundary Layer Oxygen Diffusion Limitation Hypothesis for Sea Star Wasting

Ian Hewson, Department of Microbiology, Cornell University

**I was asked recently to put together a non-oceanographer/scientist explanation of sea star wasting and our recent work on boundary layer oxygen diffusion limitation, which is below**

Seven years into our investigation of sea star wasting, we have learned much about starfish, their biology, and how they interact with their environment. After ruling out any consistently associated microbe that causes disease (i.e. a pathogen), we hypothesized that sea star wasting is not an infection (not caused by a microbe) at all. Furthermore, while there are some environmental conditions (like temperature, meteorology) that correlate with sea star wasting disease, these are not consistent between sites where sea star wasting occurred. Recent work that we performed looking at the microbiome (i.e. unicellular microorganisms associated with sea stars) and how it changes as animals waste indicated the growth of species which are unable to survive in oxygen, which led us to investigate how these low oxygen conditions occurred. In this overview, I’ll provide some background information about marine microbiology and biological oceanography, and then explain our hypothesis in non-scientific terms for the general public and for scientists who do not focus on these disciplines. First, a few definitions:

Organic Matter

Organic molecules include things like carbohydrates, proteins, amino acids, and even things like DNA and bits of cell walls. Almost all organic matter in marine habitats comes from algae, and most of the algal organic matter comes from phytoplankton (unicellular plants that float around the ocean). In coastal habitats, there can also be some organic matter that originates from terrestrial runoff (i.e. brought in from rivers, creeks, surface runoff, stormwater discharge, and groundwater), especially during ‘deluge’ events. There is also a lot of organic matter that comes into coastal areas through upwelling (which occurs when water is drawn into coastal environments by prevailing winds, etc.). Because everything is affected by gravity, organic matter sinks – so it becomes increasingly enriched as you move from surface waters into deeper waters. When upwelling happens, this deeper organic matter comes to the surface.

Why do algae produce dissolved organic matter? In microscopic plants it is because they experience ‘excess photosynthesis’ which means they fix more carbon dioxide than they can retain in cells. In larger plants, this also occurs; in kelps and other macroalgae, this also serves to lubricate fronds and keep them from breaking. You can see the organic matter in kelp forests from the bubbling and frothing that occurs on the water’s surface.

Other sources of organic matter in seawater include decaying animal carcasses, fecal matter of animals including zooplankton, and what’s called ‘sloppy feeding’ which is where animals consume most of their prey but leave some behind. Quantitatively in the ocean, these sources are typically very small, but you can imagine when there are a lot of dying organisms, or a lot of zooplankton pooping and eating, these can become a much larger proportion of available organic matter.

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Santa Cruz, California: Kelp beds here produce much organic matter, which gets whipped into a frothy ‘sea foam’ and slick on the surface

Heterotrophic Microorganisms

The ocean is a soup of bacteria, viruses, and other unicellular life. Almost all of these microorganisms (minus the viruses, which don’t eat) consume organic matter through a process called heterotrophy. Like all life, these microorganisms need materials to build new copies of themselves (i.e. replicate). When they eat and grow, most consume oxygen (i.e. they respire). In every drop of seawater, there usually 1 to 10,000,000 heterotrophic bacterial cells. When there is a lot of organic matter available, these bacteria consume a lot of available oxygen, and in doing so they deplete oxygen in seawater. This leads to ‘hypoxia’ which is functionally defined as < 20% available oxygen. When hypoxia happens in a large area, it can be called a ‘dead zone’, since animals and plants are suffocated by the lack of oxygen.

Controls on Oxygen in Seawater

Oxygen is normally saturated in coastal seawater, since oxygen readily moves from the atmosphere through the water’s surface and in bubbles (waves, etc). However, oxygen is reduced when organisms respire; this is mainly controlled by heterotrophic microorganisms. In some cases, when deeper waters are restricted in movement (in, say, basins on the ocean floor), oxygen is not replenished by atmospheric diffusion. Photosynthetic organisms produce oxygen using sunlight to fix carbon dioxide when lit by sunlight. However, they also consume oxygen at night, so there are large changes in oxygen concentrations between day and night due to what is called ‘diel cycling’. Another important physical factor controlling oxygen concentration is temperature – warmer seawater by physical chemistry can only saturate with oxygen to a lower concentration than colder seawater. In other words, the most oxic seawater is during winter, and the least oxic seawater occurs during summer.

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Bubbles caused by wave crashing generally saturate oxygen.

Some Organic Matter is Easier to Digest Than Others

Heterotrophic microorganisms consume some types of organic matter more easily than other forms. The organic matter that comes from algae is for the most part easily consumed by bacteria. Organic matter from terrestrial sources can be easy or hard to consume, depending on where it is from. For example, tea-colored material which comes from swamps and pine forests are harder to consume (we call this ‘recalcitrant’), while riverine organic matter (i.e. clearer rivers where there are lots of plants) is very easy to digest (we call this ‘labile’) by marine heterotrophic microorganisms. Decaying carcasses are generally more difficult to consume than algal-derived organic matter. Organic matter that comes up from deeper waters has been chewed on by heterotrophic microorganisms as it sinks, and becomes less and less easy to eat as it undergoes this process. Hence, organic matter brought to the surface during upwelling is not that easy to digest by heterotrophic microorganisms. However, upwelled water also contains a lot of other nutrients – which we call ‘inorganic’ nutrients-  including nitrate and phosphate.  Plants demand these for growth– so upwelling very often results in phytoplankton blooms, which in turn causes a lot of juicy organic matter to be produced.

Phytoplankon and Macroalgae

Phytoplankton (microscopic algae that are suspended in water) exist at fairly high densities even in non-bloom conditions (a bloom means you can see them with your eye). But under certain conditions algae can make the water like soup (i.e. a bloom); the causes of these blooms include enrichment with nitrogen and phosphorus (or sometimes other ‘inorganic’ nutrients), but also physical conditions (e.g. warmer temperatures or greater stratification – i.e. stability of the water column). Different species of algae have different optima for each of these stimulants. Interaction between these different factors in some water bodies results in characteristic spring and fall blooms (but sometimes there is just one summertime bloom). During winter, temperature limits growth of algae, and there is also less light; as spring approaches, this temperature limitation is alleviated, and algae rapidly take up nutrients to bloom (i.e. spring bloom); this is followed by both elevated herbivory and nutrient exhaustion; at the end of summer, as the herbivores die and nutrients become available again there can be a second bloom (i.e. fall bloom); then this dies off as waters cool and light becomes less. Summer/Fall bloom conditions are typically followed by a deficit in oxygen as the carbon fixed by plants is consumed by heterotrophic microorganisms.  Macroalgae also have a seasonal cycle, where they grow during spring, produce a bunch of organic matter at their maximum extent, then as fall kicks in they begin to degrade (being broken down by small animals and either concomitant or followed by microbial growth), and in mid-winter they are at their least extent.

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Macroalgae, like this giant kelp (Macrocystis pyrifera) produce lots of organic matter in summer, but not much in winter.

Heterotrophic Microbes are Diverse!

Bacteria are enormously diverse (something like 100,000 ‘species’ in a drop of seawater); nearly 100% do not cause any disease, but just eat organic matter and grow. Their metabolisms are mostly aerobic – in other words, they consume oxygen to ‘digest’ organic matter (this is known as terminal electron acceptor use). However, some bacteria are flexible and can use not only oxygen but other things too – like nitrate, metals, and even organic matter). These are known as ‘facultative anaerobes’. There are also bacteria that can only exist in the absence of oxygen (the oxygen is actually toxic to their metabolism). These are known as ‘strict anaerobes’. In plankton, almost all heterotrophic microorganisms are aerobes or facultative anaerobes; in deep sediments in coastal areas, almost all bacteria are either facultative or strict anaerobes. The process of taking in organic matter and converting it to carbon dioxide or other non-organic molecules is known as ‘heterotrophic remineralization’. The ‘mineralization’ part comes from the fact that organic matter is converted to inorganic (i.e. mineral) matter. A good example is breaking down glucose (which is organic matter) into carbon dioxide (which is inorganic).

Biophysics and Boundary Layers

Over the top of all surfaces in water exists what is known as a ‘diffusive boundary layer’. A good analogy is wind as it moves across land – if it’s travelling over a lawn with short grass it moves at the same speed but slows down right below the top of the grass. However, in a forest of trees, wind slows down much higher (at the canopy) and can be still near the base of the trees. The same thing happens to all surfaces in seawater – if there is a lot of terrain on the surface (for example, around rocks and pylons) water moves more slowly than if on a bare sandy bottom. This boundary layer is directly related to two things: 1) the speed of the water flow (i.e. currents, wave motion, etc); and 2) how corrugated the surface is – which is known as ‘rugosity’. Under the same flow conditions, there is a much higher boundary layer on a rugose surface compared to a completely flat surface. Because gases and other matter diffuse over distance, these boundary layers can become important to their availability at the surface. For example, within sediments there are a lot of bacteria and buried organic matter. These bacteria consume organic matter, and need Oxygen to do so – so, there is a gradient of oxygen in the diffusive boundary layer within a few mm of the sediment surface.

The surface of two species (Pisaster ochraceus – left and top right; and Pisaster giganteus – bottom right).

Sea Stars Passively Respire (i.e. They Don’t Ventilate)

Starfish, like sponges and corals, do not have gills (like fish) per se, but instead get their oxygen through their outer skin (epidermis), particularly through their tube feet and small pouches on their top surface (called papulae). Animals can sometimes actively ventilate – for example, they have gills or other structures which pump water across their respiratory surfaces. For example, clams continuously pump water through a siphon (the tube like thing that you can see on the top of the sediment) past their gills, and then out again through the siphon. Almost all starfish do not actively ventilate, but rather rely on the oxygen to come to their surfaces (some starfish have this weird ‘lung-like’ pouch that they expand and contract – notable in the slime star). Because of this, they need sufficient oxygen at their surface to survive, whereas many animals can simply breathe more heavily or frequently.

What happens when there is too much organic matter around starfish?

The boundary layer on top of starfish – which contains a lot of ‘slime’ (mucus), which is also a form of organic matter – is inhabited by heterotrophic microorganisms which probably come from surrounding seawater (remember there are more than 1 million heterotrophic bacteria per milliliter). If there is enriched organic matter in surrounding waters, or organic material sinks into this boundary layer, then these bacteria grow rapidly (those that are capable of rapid organic matter uptake are called ‘copiotrophs’) and use up oxygen. This causes depletion of oxygen immediately around animals. Because diffusion (i.e. movement by physics) of gases is limited in the boundary layer – think of it as having a much longer way for oxygen to go from saturated oxygen in seawater to the animal surface – this causes oxygen limitation (in other words, the animal can’t get enough oxygen at its surface to respire). The sources of organic matter enrichment may be multifold; for example, large algal blooms, breakdown of macroalgae at the end of the growing season, decaying animals, terrestrial runoff, etc.

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What happens when starfish die during a 48 hour Fedex shipment from Alaska. Note the water opacity and color- this is organic matter released from the carcass being chewed up by heterotrophic bacteria in seawater (filling the bags).

Evidence for an Oxygen-Sea Star Wasting Association

We observed that the types of bacteria inhabiting animal surfaces shifted from aerobes and facultative anaerobes to strict anaerobes and a few facultative anaerobes at the onset of wasting in experiments, and when comparing bacteria between healthy and wasting starfish from 2013-2014. We also saw the rapid growth of copiotrophs before wasting started. So, this told us that something stimulated bacteria (i.e. large amounts of organic matter), this caused depletion of oxygen near the animals surface to the point that anaerobic bacteria could survive.

This led us to perform a few experiments looking at several factors. First we decreased the amount of oxygen in the water column (this is done through bubbling with nitrogen gas, and we achieved a ~40% reduction in saturated oxygen which doesn’t even qualify as ‘hypoxia’) – this caused consistent wasting in the lowered oxygen treatment, but all animals in the control (bubbled with air, so full oxygen) did not waste. The bacteria on the animals surface followed the same shifts as observed during wasting progression.

Next, we did a series of experiments looking at various stressors, including water flow rate (faster flows resulted in slower wasting compared to slower flows – i.e. faster = more oxygen), animal desiccation (they wasted faster when they were left in the air for an hour compared to those which were continuously submerged), and challenge with mashed-up tissue (called a tissue homogenate) from a wasting star (more about this in a paragraph or so!), which suggested marginally faster wasting when compared to a homogenate which had been treated to remove proteins, but both these wasted far quicker than sea stars to which nothing was added.

Then, we performed an experiment in which we added several sources of organic matter, including phytoplankton concentrated from a coastal site, a cultured phytoplankton, and something called peptone, which is a protein digest and is used to grow heterotrophic marine microorganisms. All of these caused faster wasting than controls (no amendment) and again we saw the expected shifts in microbes living on their surfaces.

So, this is quite good evidence that organic matter can stimulate wasting, and that this is associated with a shift in the oxygen conditions around the animal’s surfaces.

What about viruses?

We are now 100% convinced that SSaDV, nor any virus, is associated with wasting. Here’s why. First, SSaDV is not associated quantitatively with wasting. Early in our investigation we highlighted SSaDV as weakly associated with wasting by looking at viruses occurring across multiple species and maybe found mainly in wasting-affected carcasses. The relationship from the getgo wasn’t perfect, but we used this information to then design something called quantitative PCR (qPCR) primers (currently used in COVID-19 diagnostics) to figure out whether animals with sea star wasting had more of this one virus than healthy animals. What we didn’t realize at the time was that there were many different densoviruses in sea stars which were so closely related that we couldn’t differentiate them with quantitative PCR. Once we learned more about the true diversity of densoviruses and tightened up our qPCR assay, we found no statistical association between sea star wasting and viruses.

In 2014 we performed what are called ‘challenge’ experiments where you take some fraction of tissue from a diseased animal and place it close to or inside (i.e. inject) it into healthy animals then look for the disease to appear. Normally this challenge uses purified viruses, isolated and cultured bacteria, or single compounds isolated from individuals. However, in 2014 we basically mashed up tissue, filtered it to remove everything larger than a bacterium, and then injected this into animals. The problem here is that there is much material that is smaller than a bacterium – yes, there are some viruses, but there is also a ton of organic matter – cell debris, etc.  So we cant say for sure that the responses we saw in our 2014 work were due to viruses at all. Rather, it could very well be that the organic matter that we amended into/onto seastars stimulated bacterial growth, which in turn caused hypoxic conditions. In our most recent studies, we have measured the growth of a few bacteria we isolated from the surface of starfish on sea star tissue homogenates – and their growth rates were stimulated much higher than other organic matter sources. So, we can reinterpret these studies as organic matter enrichments through the challenge caused the same boundary layer oxygen diffusion limitation effect.

Having said this, we do find an interesting phenomenon with viruses in tissues during wasting. During a study of wasting progression, we saw 35+ viruses all become super abundant as animals wasted – so something stimulates them generally. Densoviruses are a natural part of sea stars – we find them in New Zealand, China, Hong Kong, Australia, the east coast, etc. SSaDV (which is a densovirus) is likely one of the natural, non-disease causing viruses that just became really abundant in tissues late in the wasting process.

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This is a photo of the actual ‘virus-sized material’ filtered from sea star tissues used in 2014. The brown color is not viruses, but rather an assortment of organic matter. By microscopy, this contains very few virus-like particles.

Why was there mass mortality in 2013 – 2014? What changed?

This is a super difficult question to address without a time machine! 2013 was a very weird year in terms of climate for much of the area. For example, there was considerable drought in the Salish Sea catchment and in California in 2013 which correlated with wasting; and drought in 2014 in Oregon also seemed to correlate. But we see wasting without drought, and no wasting with drought at other sites. 2013 saw enormous phytoplankton blooms in the Salish Sea, but because people weren’t looking and there were no buoys active adjacent to wasting sites, it is hard to know if this occurred elsewhere. It is possible to look at chlorophyll in coastal waters using satellite imagery, but the math used to calculate algal biomass gets confused by higher sediment loads (typical of coastal regions), and like buoys are often inferred from locations well offshore. One thing we have been able to look at is citizen scientist (Jan Kocian) reports of wasting in the years from 2014 and compare these to data from a nearby databuoy at a mariculture facility. At this site, wasting occurs almost always at or just after the peak annual algal bloom; the buoy also measures oxygen, and wasting always occurs at lowest oxygen condition.

Regionally, 2013 also saw the highest upwelling ever recorded along the California coast; and along Oregon, wasting has already been correlated by researchers with lower temperature and higher acidification (both indicative of upwelling – which fuels algal blooms and thus organic matter availability).

One very cool time machine we’ve used is the ratio of stable isotopes in tissues from 2013 – 2014. Nitrogen and carbon (and most other elements) have more than one form of isotope – most Nitrogen is a lighter version called 14N, while there is also some trace amount of something called 15N. Microorganisms preferentially use 14N, which means that 15N becomes enriched as microorganisms use up the 14N. In anaerobic conditions, one form of N, nitrate, is used up by anaerobic microorganisms to form nitrogen gas (a process called denitrification); this gas is then unusable by almost all organisms. Because of this, anaerobic conditions have a very much higher amount of 15N compared to 14N. We looked at the amount of 15N and 14N in tissues of sea stars from 2013 – 2014, and found that 15N was higher in tissues of wasting-affected animals compared to healthy animals. This may be evidence that wasting animals from 2013 were surrounded by more anaerobic conditions.

We also looked at what genes were being used (‘expressed’) in wasting animals and healthy animals. One of the most important genes that all oxygen-breathing animals used is something called cytochrome oxidase; this gene comes in a few flavors, but there are some which are used in high oxygen conditions and some in low oxygen conditions only. In 2013, the cytochromes being used by wasting affected stars were all the low-oxygen ones, while in healthy animals they were both high and low oxygen cytochromes.

What about inter-species differences in wasting?

We have two pieces of evidence that boundary layer in starfish associated with wasting may affect different species in different ways. First, the rugosity of heavily affected and non-affected starfish is different – those which wasted had a significantly higher rugosity compared to those that were less or not affected by wasting. More rugose (i.e. corrugated) stars have taller boundary layers, which may limit oxygen diffusion when compared to less rugose stars.

We also measured respiration rate of several species of stars and compared these rates to the theoretical rate they could respite (which is based on physics) and found that heavily affected species were at or close to their theoretical respiration rate, while those which did not or were less affected respired a lot less than their theoretical maximum. In other words, wasted species were more sensitive to perturbations in oxygen availability than those which were less affected.

Finally, oxygen uptake by sea stars is related to the ratio between surface area (through which oxygen can diffuse) and their volume. We measured these by CT scanning, then compared the ratios between wasted and non-wasted animals.  While we didn’t see a significant difference in surface area to volume ratios between wasted and non-wasted species, smaller stars had much higher surface area to volume ratios, suggesting they may be more resilient to oxygen deficit.

 

Conclusion

We can say for sure that wasting is accompanied by a proliferation of bacteria that consume organic matter, this probably leads of limited oxygen in their immediate vicinity, and that this may trigger wasting. Wasting can be stimulated by reducing water column oxygen. We hypothesize that the sources of organic matter are phytoplankton (and maybe macroalgal), and decaying animal (resulting in a snowball effect of wasting when there are large numbers of starfish around). We also see evidence for this in both tissue stable isotopes and genetics of the animals. Something happened in 2013 – 2014 which caused a massive spike in organic matter – be it algal blooms, terrestrial runoff, or even mass mortality itself of nearby animals. Because warmer water bears less oxygen, it was and continues to be exacerbated under warmer spells, such as in late summer and fall.

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