Friday 2 February 2018

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

Tuesday 5 April 2016

Is global warming causing marine diseases to spread?

I recently attended the Oceans Sciences Meeting 2016 in New Orleans, USA (blog to come!) to present in a session called 'Scaling Up: Marine Infectious Diseases from the Molecule to the Ecosystem'. I met a group of really cool disease-y people who recently contributed to a special issue in Philosophical Transactions of the Royal Society B; ‘Marine disease’. I decided to pitch an idea to The Conversation, a news website with 'academic rigour' with the hope of gaining more attention for the important subject of marine disease.

The article, below, was a huge success, with over 6000 hits so far and was picked up by IFLScience, Science Rocks my World and the Portland Press Herald amongst other news outlets.  The Conversation believe in the free flow of information and use a Creative Commons Attribution No Derivatives licence, so you can republish our articles for free, online or in print!

Is global warming causing marine diseases to spread?

Global climate change is altering the world’s oceans in many ways. Some impacts have received wide coverage, such as shrinking Arctic sea ice, rising sea levels and ocean warming. However, as the oceans warm, marine scientists are observing other forms of damage.

My research focuses on diseases in marine ecosystems. Humans, animals and plants are all susceptible to diseases caused by bacteria, viruses, parasites and fungi. Marine diseases, however, are an emerging field.

Infectious agents have the potential to alter ocean life in many ways. Some threaten our food security by attacking important commercial species, such as salmon. Others, such as bacteria in oysters, may directly harm human health. Still others damage valuable marine ecosystems – most notably coral reefs. To anticipate these potential problems, we need a better understanding of marine diseases and how climate change affects their emergence and spread.

Warming waters promote marine diseases

Recent studies show that for some marine species diseases are spreading and increasing. Climate change may also promote the spread of infectious agents in oceans. Notably, warming water temperatures can expand these agents' ranges and introduce diseases to areas where they were previously unknown.

Many diseases of marine species are secondary opportunist infections that take advantage when a host organism is stressed by other conditions, such as changes in pH, salinity or temperature. A bacterium that is dormant (and therefore noninfective) at a certain temperature may thrive at a slightly higher temperature.

One well-documented example is the emergence of epizootic shell disease (ESD) in American lobsters. This disease, thought to be caused by bacteria, is characterized by lesions that penetrate inward from a lobster’s shell surface towards the inner flesh, making infected lobsters unmarketable. ESD can also kill lobsters by making it difficult for them to shed their shells in order to grow.

An American lobster with epizootic shell disease (ESD). para_sight/flickr

In the 1990s, following almost a decade of above-normal summer temperatures, ESD affected so many lobsters that the Atlantic States Marine Fisheries Commission declared that the Southern New England fishery (Connecticut, Massachusetts, New York and Rhode Island) was in collapse and recommended closing it. Fishery models that incorporated shell disease offered convincing evidence that ESD was a major factor in the decline of the stock. This episode underscores the importance of considering marine diseases in stock assessments and fishery management.

Now there are concerns that ESD will continue to spread north to Maine’s US$465.9 million lobster fishery. In 2015 the Gulf of Maine showed record high abundances of lobster, making it one of the most productive fisheries in the world.

However, sea surface temperatures in the Gulf of Maine have increased faster than 99 percent of the global ocean over the past decade, warming three times faster than the global average. Since temperature is a primary factor in the spread of this disease, observers fear that it could have devastating effects on Maine’s lobster fishery.

There is also a risk that ESD could spread from American lobsters to other fisheries. Seafood wholesalers have imported live American lobsters into Europe for decades, which can result in their escape into the wild. Last summer the United Kingdom’s Marine Management Organization warned U.K. fishermen that because the European lobster shares similar habitats, food sources and diseases with the American lobster, ESD could spread between the species.

As a doctoral student at Swansea University, U.K., I collaborated with the New England Aquarium in Boston, Massachusetts to investigate this possibility. While we found that European lobsters were more likely to develop shell disease when reared in the presence of American lobsters, on the positive side, they don’t seem to get the same shell disease as American lobsters.

This means that European lobsters may be better equipped to deal with outbreaks of ESD. But with sea surface temperatures in U.K. coastal waters rising since the 1980s by around 0.2-0.9 degrees Celsius per decade, it is important to monitor U.K. waters for this disease.

European lobsters with mild, none and severe shell disease. Andrew Rowley/Swansea University

Tropical disease

Now I am now studying the Panuliris argus_1 virus (PaV1) in the Caribbean spiny lobster, where the picture is more dire. Discovered around 2000, this virus is present from the Florida Keys to Venezuela. It can infect up to 60 percent of lobsters in some areas. Laboratory studies indicate that lobsters held in high-temperature seawater and exposed to PaV1 develop active and more intense infections much more quickly than those held at lower temperatures.

Studies from 1982 to 2012 show that waters in the Caribbean are warming, with the most significant temperature increase occurring over the past 15 years – approximately the period when PaV1 appeared. If PaV1 continues to spread, it could have significant effects on the health of Caribbean reefs as a whole, as well as on the valuable Caribbean lobster fishery.

Monitoring more diseases

Many other species are also showing increasing effects from marine diseases. The frequency of coral diseases has increased significantly over the last 10 years, causing widespread mortality among reef-building coral, which are home to more than 25 percent of all marine fish species.

In the Pacific, more than 20 species of sea stars were devastated by a wasting disease that ranged from Mexico all the way up to Alaska in 2013 and 2014. Research suggests that 90 percent of some populations were wiped out, and some adult populations have been reduced to a quarter of pre-outbreak numbers.

Scientists believe the cause is a virus which becomes more active in warmer conditions. In both field surveys and laboratory experiments, starfish were found to react faster to the disease in warmer water than in cooler temperatures.

Starfish on the shore at Umpqua Lighthouse State Park - Winchester Bay, Oregon. skipplitt/flickr

As the oceans continue to warm, it is crucial to understand how our actions are affecting marine life. Some species will not be able to withstand the increase in temperature. The most recent U.S. National Climate Change Assessment projects that outbreaks of marine diseases are likely to increase in frequency and severity as waters warm under climate change. Researchers are working around the world to determine whether and how species will survive disease events in our increasingly altered oceans.

The Conversation
Charlotte Eve Davies, Postdoctoral Researcher at the Institute of Marine Sciences and Limnology, Universidad Nacional Autónoma de México (UNAM)

This article was originally published on The Conversation. Read the original article.

Tuesday 1 March 2016

Mojito.... I mean... Puerto Rico!

I seem to have gone on a rampage about lobster blood over my last few posts. I will relieve you of the science with a tale of some exciting sunny marine biology!

As some of you know, as well as my research, I work at Swansea University as a part-time teaching assistant, and last year the university started it’s new Tropical Ecology Field Course, in Puerto Rico. Now, as most of you will not know, over the past year I have been in talks with a university in Mexico about applying (and indeed applying for) a postdoc. I found out in October that I got the funding for the project and I have been planning my departure from Swansea. However, I decided to go out with a bang and volunteered as a member of staff for the field course in Puerto Rico (hard life, I know). My Caribbean Sea-life knowledge wasn’t really up to scratch so I thought this would be an excellent chance to get to grips with what will probably be the next two years of my life. The trip was lead by Dr. Richard Unsworth (seagrass lover extraordinaire) and Nicole Esteban (sea turtle expert) in addition to Dr. Ed Pope (of PhD viva fame), Dr. Ian Horsfall (sea cucumber hugger) and Dr. Penny Neyland (plant fondler…. hang on, what’s she doing there?! Tehe)

So, in the early hours of a cold January morning we set off from Swansea, armed with foldable quadrats, dissection trays and bikinis (all the essentials... ya know). After a 4-hour coach to Heathrow, a flight to Houston Texas (where we may or may not have left the bags on the luggage carousel and Ed may or may not have tried to exit the airport without the students).. another flight to San Juan, and another 3-hour coach we arrived in the little town of La Parguera and our home for the two weeks; Isla Magueyes Field Station. Which by the way, was just a field station, on an island. I was picturing a larger island, with roads and stuff (as were some of the officers at the American immigration apparently, when they tried to get some students to write a street address.. another story!)… it was paradise.

There were a few iguanas.

Luckily, Rich and Nicole had been there for a few days already getting everything ready for our grand arrival (oh yeah, did I mention we were bringing 22 students as well?). We had a briefing in the classroom followed by an introductory snorkel. Although at the time not everybody was up for it, this was probably the best idea - we had been travelling all night and most of us were zombies but if left to our own devices we would have just slept and jet-lag would have ruled!

The first ‘official’ day was snorkelling practise from the various wharfs and docks around the island, with a fish measurement and biomass estimation activity, whereby we set out a line of wooden fish (lovingly transported by students last year) which we knew the size of, and the students had up to 3 tries to improve their guesses. This is really important for things like AGRRA surveys where you can use the length of a specific species to estimate it’s biomass using info freely available on FishBase. Other activities were fish ID (self explanatory), fish behaviour (trying to follow a fish for a few minutes is HARD), fish species and fish abundance, where students experienced the difficulty in estimating fish abundance underwater. 

Day three involved boat based snorkelling… now here let me introduce you to something essential that we all loved to hate. An SMB, or surface marker buoy... is, as the name suggests.. a buoy which marks the surface where a diver/snorkeller is underneath the water. In a tourist hotspot like Isla Magueyes and around, these were essential for safety.. but sometimes.. they got in the way. Now, not naming any names… but I’m pretty sure that we didn’t end the week with all the SMBs we started with (I’m looking at you, Jack.. Elizabeth…). Having an SMB entangle itself around your neck/snorkel/weight-belt, let me tell you, is not a nice experience... but neither is Richards face when you have to tell him you tried to tie one to a rock then lost it! Anyway, SMBs aside.. today we used the carefully re-assembled quadrats to look at percentage cover of corals, sponges, algae, seagrass (because nope, they are not the same thing) plus the invertebrates on the seabed.. in both the day and the night (spooky!!). This activity essentially taught me how bad my coral ID skills were but hey! I had another week to improve. And to play with the territorial damselfish... 

The next couple of days were based on teaching and learning AGRRA. AGRRA, or to use it’s full name; Atlantic and Gulf Rapid Reef Assessment is a technique used to assess coral reef benthos. This includes understanding how to assess coral reef health, and for our students, to assess coral reef health of reefs in Puerto Rico by examining how the biota of healthy reefs changes as they become degraded. There is also a technique for assessing coral reef fish assemblages, which applies the knowledge of reef fish we developed on day 1 to assess coral reef fish communities in Puerto Rico. Again, we used it to examine how the fish communities of healthy reefs change as they become degraded. Now obviously, our surveys were small, but when used for research projects, these techniques are widely comparable and are used by scientists in Universities, Government and NGO’s for assessing coral reef health in Caribbean and Pacific.
Early morning commute to the sampling sites. Life is hard.

Even though it was a marine ecology field course, a super important part of tropical marine ecosystems are mangroves. So, under the supervision of our resident plant lover Penny, we headed out to Laguna Monsio José to learn about these fascinating ecosystems.. because yes, although a mangrove tree is a plant, the forests mangroves form are among the most productive and biologically complex ecosystems on Earth. As described in this great NatGeo article.. “birds roost in the canopy, shellfish attach themselves to the roots, and snakes and crocodiles come to hunt. Mangroves provide nursery grounds for fish; a food source for monkeys, deer, tree-climbing crabs, and a nectar source for bats and honeybees”. As well as squelching through the mangrove mud, we snorkelled through the roots to check out diversity of fish that live there... maybe plants are pretty cool after all.

The students also learnt how to seine net.. and this was an interesting one. As marine biologists, they are lucky in the fact that they have already taken part in a field course in the UK (at the Field Centre in Orielton) so are familiar with netted species back home.. so here we did it at night and in the morning. Both of which I missed as I was asleep very busy science-ing. 

Now.. we also did a lionfish dissection. In the Caribbean, the lionfish (Pterois volitans) is invasive. That means, it's not supposed to be there. Native to the Indian Ocean, Southern and Western Pacific Ocean and the Red Sea, it is speculated that they were introduced to the Atlantic when released by "retired" aquarium enthusiasts. Luckily, cold water temperatures are keeping numbers at bay in the north, but this is not the case in the south where lionfish are spreading rapidly through the South Florida coast, the Gulf of Mexico and the Caribbean Sea. But so what? They are just fish right? Wrong. Lionfish are are voracious predators and non-selective feeders, with virtually no natural enemies due to their toxic spines. Studies have shown that a single lionfish can reduce juvenile fish populations by 79% in just 5 weeks. Wow.

We found some pretty cool stuff in our lion fish stomachs... including a mantis shrimp!!

Mantis shrimp! Fresh from a lionfish tummy
My favourite day by far had to be the seagrass sampling.  SeagrassWatch is the internationally recognised method for assessing seagrass meadows. It allows scientists to examine the differences between healthy and degraded seagrass meadows and our students were able to help establish a long-term seagrass monitoring site in Puerto Rico! If you are a marine scientist that is interested in taking part.. check out the manual here

Seagrass are important; like mangroves, they support whole ecosystems. The habitat complexity within seagrass meadows enhances the diversity and abundance of animals. Seagrasses on reef flats and near estuaries are also nutrient sinks, buffering or filtering nutrient and chemical inputs to the marine environment.... They also stabilise coastal sediments. Most important of all, they are a nursery for all sorts of reef critters... including my buddy, the spiny lobster. Below is a video of me doing what I do best, harassing a couple.

The last few days were reserved for the students to undertake their very own 'mini research projects'. These 5 projects ranged from tarpon behaviour... to abiotic driver of benthic composition, the latter of which I was lucky enough to take part in! 

Overall, a great week was had by all. I can say that although I went as a member of staff, I was constantly learning and I feel safe in the knowledge that I now know my squirrelfish from my angelfish. A must, if you plan on undertaking a postdoc in the Caribbean... (but more of that in my next post!)

Tuesday 16 February 2016

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! 

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!

Friday 11 September 2015

Team lobster! A meeting at the National Lobster Hatchery

The National Lobster Hatchery, Padstow
Last Friday, I was asked to attend a meeting at the National Lobster Hatchery in Padstow, Cornwall. I know I've blogged about it in the past, but collaboration with other research groups is, to me, one of the most important factors when conducting a project. As well as a hub for lobster science in the UK, the national lobster hatchery has great ties with the local fishermen, the Padstow community and restaurants in the area, with their very successful 'buy one set one free' campaign.

I've liaised with Padstow in the past - when I had visiting researchers in my laboratory from the New England Aquarium, Boston, this was on our list of places to visit; and when I needed juvenile lobsters for exposure studies, I would contact the hatchery. It was only at the 10th International Conferenceon Lobster Biology and Management, that I really got to know the 'hatchery lot' and we became great friends, and colleagues. We vowed that we would stay in touch and try to meet as often as possible in order to discuss the future of European lobster research in the UK.

In attendance at the meeting on Friday were hatchery staff Dom Boothroyd, the general manager; Research & Development Officer Dr Carly Daniels; Business Development Officer, Clare Stanley and PhD student Charlie Ellis, who is part of the University of Exeter's Falmouth Campus, but works closely with the hatchery on his research project. 

A lobster with one of Dans tags on it's 'arm' 
First up to talk was Dr. Daniel Skerritt, who completed his PhD at Newcastle University last year, investigating lobster abundance and movements in Northumberland. Dan now works as a consultant for MRAG in London and gave us a talk about his research findings both during and post- PhD. For his project, Dan monitored lobster behaviour in and around baited pots (used to catch lobsters), and their interactions with habitat using acoustic telemetry. Perhaps his most significant findings which may have the greatest implication to management, concern differences between the sexes. From mark-recapture studies (where a lobster is tagged, released, and caught again) he found that males have a much higher catchability than females. This means that a lot more male lobsters were recaptured – but why? The acoustic telemetry work revealed further differences between the sexes; males use a much larger area of seafloor than females, which could account for this increased catchability due to greater probability of pot-interaction. However, overall this work focused on the utilisation and behavioural changes over substrate. Dan has a publication in press for Marine Ecology Progress Series; “Fine-scale movement, activity patterns and home-ranges of European lobster Homarus gammarus the prepress abstract can be viewed here.

Aside from his science, Dan has also been involved in some outreach work. He struck up an interesting collaboration with a graphic designer and the Great North Museum. They put on an exhibition with input from Natural History Museum called ‘Spineless’, with Dan’s work being the subject of one of the exhibits. The aim of the collaboration was to make the kids of the northeast aware of the importance of the lobster fishery; you can see more about the exhibit here.

Check out this great little video of Dan, talking about his research.

A snippet from my lecture 
Up next, I gave a talk about the main findings from my PhD; I have talked mainly at conferences about my shell disease susceptibility work but my lesser known research concerning parasites (see last weeks blog post... and more in next weeks!) and MPAs, were very interesting to share. It's great to talk informally about this, and to get some ideas together for future work. 

Charlie is currently writing up his PhD and gave us a short overview of his findings so far. The National Lobster Hatchery's main mission is to create a sustainable lobster fishery in Cornwall and in order to do this, the number one research priority is to monitor the success of it's primary charitable objective (i.e. the stock enhancement program). In order to do this, they must be able to estimate survival rates for hatchery reared lobsters in the wild, as well as their contribution to catches of landing-sized European lobster. To do this, genetic analysis of Cornish lobster stocks is essential, and something that Charlie has been working on. He has also been examining tagging systems that will enable stakeholders to easily identify hatchery reared animals. So far, Charlie has found that the lobsters around the Cornish coast all seem to come from one gene pool, which is good for the release programme which relies on volunteers to bring in berried hens (expectant lobster mums) from various locations. 

Spot the baby lobsters!
I think we are a very talkative lot so we didn't have much time for poor Carly to talk to us about her new and exciting project which focuses on developing sea based culture of lobsters in containers, a rearing technique that exhibits the potential for a low carbon form of rearing with no feed costs. This is a consortium project, led by the National Lobster Hatchery, which follows on from an earlier project also funnded by Innovate UK/BBSRC. Carly completed both her BSc and PhD projects at the hatchery, concentrating on the optimisation of the rearing diets for early life stages of the European lobster, in order to enhance growth, survival and health using biotic dietary supplements.

The hatchery also hosts students who work on small but important projects and so we also heard interesting presentations from Dan Sankey, who is working on lobster behaviour and is soon to begin an MRes at Swansea University; and Grace Dugdale, a BSc student at Cardiff University who is working on a placement year alongside Carly at the hatchery. Grace is looking into the effects of probiotics on lobster juveniles. Also in attendance were Adam Bates, who is working towards an MPhil in European lobster genomics and Joe Augier who previously completed his undergraduate project at the hatchery and is going on to do an MRes.

In all, it was a great way to reconnect with the lobster team, over a year after meeting at the ICWL in Mexico. I would like to acknowledge all in #Teamlobster for helping me to write this blog post… lobster scientists, unite!

Monday 31 August 2015

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 1931H. 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