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!

Fisheries, management and guest-blogging!

Just a quick one today really... If you are a close friend or follow me on twitter you will have seen me harping on about a guest-blog I have recently written for the Society of Biology. The SOB were one of the funders for my trip to Canada and the USA back in October - they gave me £500 and for this, I had to write a report on what I got up to whilst there, which you can read here. Whilst I was there, they noticed that I got some press coverage and saw that I had a blog, so I was asked me to write a guest-blog on my experience. I've been so busy that it took me months, so finally it was published last week. I'd like to thank Dr. Andy Woolmer for his help with the article.

Obviously, the main reason I went to Canada was to learn new techniques and more information about lobster diseases such as gaffkaemia (see my first blog), but since I am still working on the analysis and results for that, I decided to take a different approach when writing my article. I am fascinated by fisheries, sustainability and conservation and hope to one day work in fisheries management of some sort.

Currently, there is a review of all fisheries legislation in Wales, which commenced in January 2012 and whilst over in Charlottetown and Boston, I had some discussions regarding local laws and fisheries management, some of which were really different to ours, so I decided to talk a little about how we manage things over here, compared to how things are done over there, where awards have been won. If you'd like to go for a read and learn a little more about Welsh vs. North American lobster fisheries (and see some pictures!) click here, or.... I have copied and pasted the whole article here:

'What we can learn from our peers around the globe?

Guest blogger Charlotte Eve Davies, a PhD student at Swansea University, talks about receiving a Society of Biology Travel Grant to go to the AVC Lobster Science Centre, Canada.

‘So what do you do?’ is the question I get asked rather often. People look at me and assume, at the age of 24, I should be settled down with a ‘grown up’ job. Alas, I am still studying, but I wouldn’t have it any other way.

In truth, there isn’t a single word to summarise my work. Underpinned by a degree in biology, I have since branched out into various areas. Pathologist? Maybe. Marine biologist? I like to think so. Lobsterologist? If only that was a word! I like to keep my options open.

Last autumn, with the help of a Society of Biology Travel Grant, plus one from the Climate Change Consortium for Wales, I was able to take my love of all things lobster to the Canadian province of Prince Edward Island.

During my time ‘across the pond’, I learnt a lot about lobster fisheries!

When you’re looking at my area of research – assessing lobster disease, fisheries are the target. I think that Welsh fisheries management could learn some valuable lessons from the systems implemented in the US / Canada. Over there, v-notching, a system where females with eggs have their tails cut to indicate their ability to produce offspring, thus enhancing future stocks, is mandatory. Both scientists and fishermen alike there recoiled in horror when I told them that in Wales you can still catch and land berried hens (egg carrying females).

In Maine, USA, 1872, the first law was implemented banning the capture of berried lobsters, but it was a measure already practiced by many Maine lobstermen. Last year the Maine lobster fishery was awarded Marine Stewardship Council (MSC) certification as a sustainable and well-managed fishery. The Prince Edward Island lobster fishery entered into the process of being assessed for the same award whilst I was there.

Why are we so behind the times?

Unlike the co-managed Inshore Fisheries and Conservation Authorities in England, fisheries in Wales are managed by various sectors of the Welsh Fishermen’s Association, who decide upon Sea Fisheries Legislation, or ‘by-laws’, which are then implemented by the Welsh Assembly Government.

Last year (2013), the Llŷn Fishermen’s Association bravely decided to vote in favour of implementing their own voluntary ‘berried ban,’ – disallowing landing of females with eggs. This unilateral move may have influenced the recent decision by the Welsh Government to propose a berried ban.  Amongst a raft of new crustacean management regulations being proposed, they are consulting on a berried ban in Wales for the long term benefit of Welsh fisheries.

The current Welsh crustacean consultation includes an evidence report outlining the case for a ban and other potential best practice management measures aimed at securing the long-term sustainability and profitability of the Welsh fishery. In Wales, unlike the rest of the UK, fisheries regulations are able to extend out to the 12 mile limit which really makes these effective management measures.

Opposition to these proposed by-laws comes from some fishermen, who worry about a depleted catch if the berried hens are off limits. However, it has been found that putting berried hens back does not cost the fishermen anything after the first season and the lobsters put back today can be recaptured once the eggs are shed.

Each 90mm lobster returned produces 7 lobsters for the fishery – based upon documented egg production at that size and assuming only a 0.1% of eggs result in lobsters entering the fishery. It’s a lobster win-win!

American and Canadian lobster fisheries are a lot larger than ours, but considering their success, we can afford to take some tips from them. The UK landed more than £32m worth of lobster in 2011, but unfortunately there are also imports of American lobsters into the UK (mainly for the restaurant industry), introducing an ‘invasive species’, leading to hybrid Euro-American lobsters and the possibility of disease transfer… but that’s a whole different kettle of fish!

Before I leave, a few words of advice on the collaboration front. Never give up. If you don’t ask, you don’t get, and if you have an idea, make it heard! You never know, you could wind up halfway across the world kissing seals (yuck), feeding turtles, and learning an awful lot more than you bargained for…

For more of my ramblings, check out my blog and find out more about the proposals for the inshore crustacean fishery.'

Whilst I know for many PhD students that writing their thesis and getting papers published is the main priority, I believe that writing small things like this, that are interesting to the public when written in an informal manner, are also a really good way of raising the profile of your research as well as getting those who wouldn't usually be interested in science, interested!

I should also mention that the Society of Biology offer £500 Travel Grants to student affiliate/AMSB and Early career/MSB members, and the deadline for the next round of grants is 31st March 2014. I get lots of people ask me about how I self-fund my PhD, so I will be writing a blog soon about funding opportunities, and my experience applying for them, so stay tuned!