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! 

Lobster blood suckers and the wonders of histology

So I promised a couple of blogs back to write a post about some of my lesser known work on parasites. If you have read some of my oldest blogposts, you will know that I entered into the world of lobster-loving through my undergraduate dissertation (or final year project, as some universities call it).

My dissertation focussed on a little known parasite Nicothöe astaci, otherwise known as the lobster louse. A parasite which lives on, and feeds on blood from, the gills of the European lobster. Now, this little critter has been documented for well over 100 years as it was first noted in 1826 by Audoin & Milne-Edwards. It has been found only on European lobsters but ranges from those inhabiting locations including Scotland, Lundy Island in the Bristol Channel and as far south  as Portugal. It has since only been written about a handful of times, and before my dissertation, the last work was over 50 years previous in 1959!

I was tasked with finding out exactly how the parasite attaches to the host, using a technique called histology. Now, histology, the study of the microscopic anatomy of cells and tissues of plants and animals, is a useful technique and one of my favourites. It is used in a science called histopathology, the microscopic study of diseased tissue, and is an important tool in pathology, since accurate diagnosis of diseases usually requires histopathological examination of samples. Histology first requires the samples (be it tissues, or whole parasites) to be embedded in a paraffin wax block, which is then sectioned into very thin slices (up to 10 microns thick!) using a machine called a microtome. Theses slices are then fixed onto microscope slides (I use albumin-glycerol) and left to dry before being stained.

I use Hemotoxylin- Eosin staining (sometimes called H&E stain) a common stain used in medical diagnosis. Hematoxylin is dark blue/violet which is basic/positive which binds to basophilic substances like DNA/RNA (which are acidic and negatively charged). Therefore things like the nucleus, ribosomes in the rough endoplasmic reticulum, and sperm cells are stained violet/blue. Eosin is a red/pink stain that is Acidic / Negative and so binds to acidophilic substances such as positively charged amino acid chains which make up proteins.  Therefore, things like cytoplasm, muscle cells, intracellular membranes, and extracellular fibers are stained pink. 

Finally, a coverslip is glued on using a mountant called DPX so that the scientist can look at the slide using a microscope. Cool hey!

Photograph showing an example of histological preparation. The paraffin wax block containing the sample (P) is being cut using a microtome. The thin slices (S) are then placed on a slide before staining and mounting. Photograph edited from original.

Before I could look down my microscope for this all important point of attachment... we had a few problems. The Nicothöe parasite is a copepod, and copepods are a group of around 12000 planktonic species of the phylum Crustacea (that's the same as a lobster... i.e. it has a hard shell!). This meant that when we were embedding the little critters for histology.. we had to come up with a whole range of trial and error techniques, to stop them popping out of the wax, and ruining the blades on the microtome! We tried decalcification, cutting open the egg sacs the get the wax to infiltrate quicker, mixing Xylene into the ethanol during processing and even soaking the finished wax blocks in Mollifex™.  After a few weeks and LOTS of histology, we got the cut just right, and were amazed to find the point of attachment. I was exhilarated by the science, by finding something new, that nobody had ever seen and by working hard to get to that point (a scientist was born!).  My first publication came from this work and even though I was only fifth author.. it was the best feeling.

Histological sections showing attachment and invasion of gill filaments by Nicothoë astaci. (A) shows attachment of N. astaci to a gill filament (G) showing the invasive feeding channel (*) through the gill cuticle.

 (B) shows Funnel-shaped feeding channel through thickened gill filament cuticle (GC) with dashed arrow indicating direction of blood flow from gill filament into the parasite. 

(C) shows  the imprint of N. astaci suctorial disc on the surface of a gill filament. Imprint of setule-like fringe (*) is also visible. Scale bars=50 μm (A, B) and 10 μm (C).
This photo is taken from my first paper available here.

Fast forward a few years to when I was a PhD student, and this little critter kept popping up in every wild lobster we sampled. Most scientists I talked to didn't think they were anything to worry about. - just harmless guys hitching a ride. I disagreed. One day, one of my laboratory lobsters moulted and I happened to catch it before it could feast on the shell. I took a fragment of the moulted gills with parasites still attached and put it under a dissecting microscope. You could see the movement of the parasites stomach, almost like the peristaltic movement of the intestines you learn about in school. It got me thinking - we knew these parasites were hematophagous (they feast on lobster blood, hence their prime position on the haemolymph-rich lobster gills) so there must be something they are doing to the host... be it good or bad.

I had read papers and news articles in the past about sea lice found in the mouths of lobsters, gills of fish and in turtles that often end in death which in turn can affect whole fisheries. It is thought that approximately 50% of copepod species live in symbiotic associations (including parasitism) with a broad spectrum of aquatic animals, ranging from sponges to marine mammals. I wanted to know exactly what these parasites were doing to the lobster. I got an email from a guy at the Ifremer Institut in Brest, France, who was in charge of stock assessment of large crustaceans such as the European lobster. He told me that he had read the paper from 2011 and thought that mortalities in the holding facility were due to high levels of Nicothöe infestation. He said that as mortality steadily increased, the prevalence of the parasite and the infestation level seemed to increase too. Interesting. Check out my next blog to find out how we went about exploring the effects of these fascinating parasites on their lobster hosts!

Crabs, parasites and other wonderful afflictions

So it's been a while since my last blog post... I know! Since finishing my PhD in January it's been a hectic 6 months. I have been busy writing up some bits and bobs from my thesis which weren't quite published.. and you will all be excited to hear that my research has moved a little towards the crabby side... (groan!).

For one of my PhD chapters, I looked at a disease called Hematodinium. Well, more of a parasite than a disease, this dinoflagellate infects over 40 species of decapod crustaceans worldwide. But not lobsters of the clawed kind, apparently.... I set out to test this theory.

So a bit of background. What is a parasite? According to the dictionary; "noun an organism which lives in or on another organism (its host) and benefits by deriving nutrients at the other's expense." There are different types of parasites, endo (those that live within an organism) and ecto (those which live outside of one). An example of an ectoparasite, is the 'lobster louse'; endoparasitic copepod Nicothoe astaci, another critter I have worked extensively on and may have mentioned in the past. In my lab, we have worked on it's histological morphology, revealing the point of attachment to the lobster, surface morphology revealing the attachment mechanism and the effects of the parasite upon the host.

Anyway, back to the parasite at hand. As an endoparasite, Hematodinium live inside the host, specifically in the haemolymph (blood)... pretty grim I know. A couple of French scientists Chatton and Poisson first reported the disease in France in both harbour Liocarcinus depurator and shore crabs Carcinus maenas in the 1930s. It has since been found to infect over 40 species of decapod crustaceans worldwide, and because infected animals become unmarketable due to poor muscle quality, Hematodinium spp. infections have had huge economic impacts on commercial fisheries. For example, in France, the velvet swimming crab Necora puber fishery suffered a catastrophic collapse (>96 %) due to Hematodinium spp. in 1985. In the US, outbreaks of Hematodinium spp. have infected up to a third of the Tanner crab Chionoecetes bairdi and snow crab Chionoecetes opilio stocks in southeast Alaska and Newfoundland respectively and in Virginia, loss to the blue crab Callinectes sapidus fishery is estimated to be between 0.5 and 1 million USD per year. In the UK, the Scottish Nephrops fishery also loses approximately £2-4 GBP million annually due to Hematodinium spp. infection.

There are only two species of Hematodinium that have been described so far. This is due to their lack of distinct characteristics and poorly understood life cycles. The type species, Hematodinium perezi, was first described from the crabs on the Normandy and Mediterranean coast of France by our friends Chatton & Poisson in 1931. H. perezi, or a closely related species, has since been reported in epidemics from edible/brown crabs Cancer pagurus and velvet swimming crabs off Brittany, France, and from the English Channel. A second species, H. australis, was described from Australia and was separated from H. perezi on the basis of size of the vegetative stage (called a trophont), the presence of rounded plasmodial stages and the austral location.

My experiment, in a nutshell

So, why do I want to see if my beloved European lobsters are susceptible to infection? Judging by the above effects upon fisheries worldwide, it's an important critter to keep an eye on, and since it infects our native Cancer pagurus (edible, or brown crab), for me, that's a little too close for comfort! Edible crabs share habitats with European lobsters and are often found together in parlour pots (fishing traps) - often injured from some aggressive run ins. We know from my past research that injury can lead to disease and although Hematodinium infections have been found more in juvenile crabs, it is still an important issue. We don't know where the parasite resides before it enters the host, and so it is interesting to investigate the susceptibility of different species in order to further understand the infectivity.

In order to do this, we did two experiments, or 'exposure studies'.  First, we collected some edible crabs from the South Wales coast, from spots known to harbour Hematodinium infected crabs in the past, and inspected the blood for the parasite. Just to be sure, we kept them for a few weeks, checking every week for infective stage parasites. Once we were happy we had some crabs sufficiently 'infested' enough, we took live samples of Hematodinium by drawing the blood (haemolymph) and separating out the parasites into a clean saline solution. This solution was to be injected into our disease-free, juvenile European lobsters.

We first did a preliminary, or pilot, study, which was run side-by-side with a similar study artificially infecting edible crabs Cancer pagurus (just to be sure that the Hematodinium species we were injecting was viable). In the pilot, the crabs injected became infected after a matter of weeks, but the lobsters did not... However, the number of lobsters we used was small and we wanted to run a longer study with more sampling points, so we decided to try again. On the second attempt we took blood samples from the experimental (and control!) lobsters before injection, just after, 24h after, 1 week and then every month thereafter. The results were as expected... all negative (even the 24h post injection one!). To look for the parasites, we used microscopy (blood smears), polymerase chain reaction (PCR) with primers specific for Hematodinium spp. (yes, that's species, just in case!) and also histology from the final time point.

What is it that a lobster has and a crab doesn't? There have been some pretty cool molecular studies of late at a collaborators lab in Canada, looking at gene expression (i.e. what genes are expressed in disease animals vs. those which aren't diseased...) I think it would be really interesting to find out exactly what it is in the lobster immune response which renders it unable to maintain this infection.

Although we weren't surprised at our results, it is still an interesting study. It does seem that the EU lobster has something that other decapods don't. Another example is my earlier disease work where we looked at transmission of epizootic shell disease (ESD) from American lobsters into European ones... to no avail. It seems EU lobsters are the strong men of the lobster kingdom?

To read the full study, see the citation below (if you click the DOI, it will take you to a download page). If you can't access the papers, feel free to comment or email me and I can send you a copy.

Davies, C.E. and Rowley, A.F. (2015) Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. Journal of Invertebrate Pathology 127, 6-10 doi: 10.1016/j.jip.2015.02.004

For further reading, my supervisor recently wrote a mini review on this interesting parasite...

Rowley, A.F., Smith, A.L. and Davies, C.E. (2015) How does the dinoflagellate parasite, Hematodinium outsmart the immune system of its crustacean hosts? PLOS Pathogens 11 (5), e1004724 doi: 10.1371/journal.ppat.1004724