Sunday 10 January 2016

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 Nicothöparasite 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 lobstersgills 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 Nicothöinfestation. 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!

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