Is lobster blood blue?

An interesting side-product of some of my experiments have been the varying colours of the lobster haemolymph. I remember once watching QI and there was a question about lobster (or was it crab?) blood - and the 'correct' answer was that it was blue. But is this correct? I remember shouting at the TV at the time that it was wrong (I was a misunderstood lobsterologist, after all...)

When I was doing my PhD, I uploaded a photo to twitter which people went a little crazy for. It was from the experiments I talked about in my previous blogpost and it even surprised me. Each blood sample was a different colour! All the lobsters were from a similar location (Ilfracombe, Devon), similar size (92 - 100cm in carapace length) and mostly female.  So, why, then, is there such a discrepancy... and most important of all... why aren't any blue?!!

Lobster blood samples for protein and metabolite quantification - the white bits are haemocyte pellets. Photo taken by Charlotte Eve Davies. 

First of all, let's get something straight. Lobsters don't have blood. I know I say it above and I will say it again, but as previously explained, lobsters don't really have blood. In fact, lobsters don't even have a 'closed' circulatory system like us (or mammals). Although they have a heart, which beats, and arteries, through which haemolymph (lobster 'blood') is passed through to bathe the organs, there are no veins to pass the haemolymph back to the heart. Instead, it returns to the heart via interconnecting spaces known as venous sinuses. For this reason, you will sometimes hear that the lobster's circulatory system known as an "open" circulatory system.

Okay, now that's out of the way, back to blue blood business. Some of us may have seen the (in)famous video or photos of bottles of horseshoe crab blood being decanted in a factory, for use in medical research... and we are wondering why my photo above doesn't show a similar brilliant blue hue? This is because, firstly, my samples were taken directly from a syringe, into a tube, which was closed, centrifuged and put in the freezer almost right away to stop the blood from clotting (the little white pellets you see are blood cells, or haemocytes, from the centrifugation). So the samples weren't exposed to much 'air' or namely, oxygen. 

As mentioned in one of my previous blogs, haemocyanins are the crustacean version of our haemoglobin; they are proteins that bind and transport oxygen throughout the body. Haemocyanins contain two copper atoms that bind a single oxygen molecule (remember it's O2... not O1) rather than Iron, which is what binds to oxygen in human and most mammalian bodies. Now, it's oxygenation that causes a colour change between the colourless Cu(I) deoxygenated form and the blue Cu(II) oxygenated form which is why in some cases, a nice blue colour can occur, especially when exposed to the open air - air contains oxygen which reacts with the copper present in the blood, giving us the blue colour.

However, this is not always the case. Protein levels in the blood, or haemolymph, of lobsters, and all crustaceans, are constantly changing. This can depend on the stage in the moult cycle (i.e. is it about to shed it's shell to grow?), the reproductive status (whether it is about to try and find a mate, or lay eggs) or even whether a lobster is diseased or not. It was interesting that in my photo, one sample was very dark green (bottom row, 4th from the right) - we think that this may have been due to reabsorption of eggs. There is a protein, vitellogenin which is synthesized by the ovarian tissues in lobsters. During female maturation, extra-ovarian vitellogenin is transported through the hemolymph to the ovary and is taken up into the cytoplasm oocytes, or eggs. Egg reabsorption can happen for a umber of reasons, be it adverse conditions (if the lobster is stressed), or if the egg were released too early and the lobster then needs to moult. 

More recerntly, during my first postdoc, I have been working on a virus in the Caribbean spiny lobster Panulirus argus. The virus, Panulirus argus Virus 1 (PaV1) has some interesting effects on the haemolymph of infected hosts. It turns it a very white, milky colour, rather than the usual amber colour, due to the degradation of hemocytes (blood cells). This is how we diagnose clinically infected individuals, pulling the tail away from the body a little and looking at the almost clear membrane covering the abdomen, it can clearly be seen whether or not an animal is infected.

Diseased vs. Healthy Comparison: The lobster on the left in this shot is healthy while the one on the right is in the last stages of PaV1. During this time the hemolymph turns from clear/amber to white, as you can see in the middle syringes. Photo from http://www.pav1.org/

So that's how we know... lobster blood isn't always blue.

Lobster blood chemistry, and gruesome infestations.

I started talking about a little parasite in my previous blog, Nicothöe astaci. I realised it was getting a bit long so decided to split it into two - the first about histology of infected animals, and another, this, about physiological effects of the parasite on my lobster hosts!

So, after hearing more about this fascinating creature I wanted to know what it did and whether the parasite load, like the French scientist had mentioned, had an effect on the physiology or even the life, of the host...

I set off to Ilfracombe and Lundy, a place we had sampled before and knew for sure that there were pretty high levels of Nicothöe. It was here that the fishermen had pointed out the parasites to our research group in the first place! I joined forces with our favourite lobster fisherman Geoff and came back to Swansea with 18 lobsters (about 10 kilos) from various points around the Ilfracombe and Lundy coast. I let them acclimate for a few weeks in the aquarium to get used to the conditions before starting any experiments. Lots of things can stress a lobster out, including being caught in a lobster pot, handling and transportation so it's always good to do this when working with live animals from the wild.

As you can see from below, we had quite a range of parasite loads on our lobsters. It ranged from just a few to alot - infestation!!!

Photographs showing examples of (A,B) low and (C,D) high levels of Nicothoë astaci (arrows) in the gills of European lobster before (A,C) and after (B,D) excision. Inset shows the structure of the parasites. Note the high numbers of parasites at the base of the gills in the lobster with high parasite load (arrows). The excised gills show the arrangement of gills into outer, middle and inner sets. Photo taken from Davies et al. (2015)

A good way to test levels of stress or changes in a lobster (or any crustacean) physiology is by testing for changes in the composition of blood, or haemolymph. This was especially true in our case, since Nicothöe astaci is haematophagous, or blood sucking! I decided to test our lobster blood for 4 key components; haemocyanin, ammonia, glucose and total protein. Haemocyanins (sometimes spelled hemocyanin) are the crustacean version of our haemoglobin; proteins that transport oxygen throughout the body. Haemocyanins  contain two copper atoms that bind a single oxygen molecule (remember it's O2) and the reason that you hear many people saying that lobster/crab blood is blue (this is not strictly true - more of this later!). Unlike the haemoglobin in red blood cells found in vertebrates, haemocyanins are not bound to blood cells but are instead suspended directly in the haemolymph. 

We also tested for total haemolymph protein - this is because haemocyanins are not just oxygen carriers. They make up approximate 80-90% of total haemolymph protein (although this changes depending on whose papers you read!) and are an important component in some invertebrate immune systems. In arthropods (crabs, lobsters etc.) the haemocyanin family includes phenoloxidases, hexamerins, pseudohemocyanins or cryptocyanins and (dipteran) hexamerin receptors. Phenoloxidase are copper containing tyrosinases,  proteins involved in the process of sclerotization of arthropod cuticle, wound healing, and humoral immune defenses. For me, testing for haemocyanin is a win-win, not only are our parasites located on the gills, where key oxygen exchange occurs, but then suck the blood, so we hoped that testing for this would give us some answers. Questions here were:

1. Does the presence of the parasite hinder oxygen transfer across the gills?

2. Does the blood sucking activity of the parasite deplete oxygen levels in the haemolymph?

3. Does the presence of the parasite deplete haemocyanin (i.e. is the % of haemocyanin in total protein higher or lower than averages)

Aquatic crustaceans excrete the nitrogen derived from protein and amino acid catabolism primarily through the gills, the gut and the antennal/green glands. Nitrogenous waste in lobsters is made up of urea, ammonia and amino acid compounds; the major excretory product is ammonia. The concentration of this waste in the haemolymph changes in response to stress and ecdysis wherefore we tested for changes in ammonia levels.

Questions included:
1. Does the presence of the parasite hinder ammonia excretion?

2. Is the presence of the parasite increasing stress-induced ammonia levels?

Finally, we tested for glucose. Glucose levels have been shown to change in line with lobster stress levels and we thought it might be affected by the parasites attaching to the gills.

So, what did we find? Safe so say, as expected, there was a positive correlation with the amount of parasites and total protein. This means that as the number of parasites on a lobster increases, so does the amount of protein in the blood. Sounds weird, until you see that the haemocyanin also increases, and it makes up 84% of the total protein in the haemolymph we tested. So, the real story here is an increase in haemocyanin as parasite load increases. We think this is the lobster most likely compensating for reduced respiratory function due to gill damage caused by the parasite. Increased haemocyanin, may therefore be advantageous for infected lobsters.

There was also a slight, but not significant, correlation with ammonia and glucose (see figure below). It could be that ammonia and glucose are not really affected by the parasites, or, as in another study, parasites can absorb glucose from the haemolymph, thereby forcing the host to resupply tissues with this sugar from glycogen reserves in the hepatopancreas in order to maintain carbohydrate homeostasis. As for the ammonia, some studies have shown a switch in nitrogenous wastes to products such as urate or urea... which we didn't test for.

This figure, taken from my paper Davies et al. (2015) shows the results of a Spearman’s correlation coefficient analysis.  You can see correlations between parasite numbers and haemolymph concentrations of (A) total protein (p = 0.02), (B) haemocyanin (p = 0.0065), (C) glucose (p = 0.2112) and (D) ammonia (p = 0.1290). Asterisks denote significance. 

As always, you can email me, tweet me, or add me on LinkedIn. I am happy to send over copies of my papers or answer questions!