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!