The SOI Early Career Network, October talks and upcoming Isle of May field season

Pregnant female grey seals and yearlings hauled out on the rocks around the Isle of May, Scotland

It’s that time of year once again, autumn is here and that means I’m making inventories and packing equipment for the PHATS team’s field season on the Isle of May. We’ll be heading out to the island at the end of October to begin our last data collection season for the project, and we’ll be living on the island and studying the grey seals until mid December. Before we head out though I’ve got a busy month ahead of me, as I’ll be presenting PHATS work, my PhD work on oxytocin and talking to the public about grey seals. But before we get onto where and when I’ll be presenting, I’m quickly going to give a shout out to a new group I’ve been involved with setting up over the last few months, the SOI Early Careers Network.

This grew out of a group of friends from the Scottish Ocean’s Institute (SOI) meeting to help each other practise for presentations, to give feedback on each other’s ideas and to chat and share resources about the various issues early career scientists face. We then decided to open the gatherings up to any early career researcher at the SOI, and the group has grown ever since. We meet at least every week, sometimes more, to discuss anything our members currently need help or advice with. Right now we are having lots of conference poster and talk preparation sessions with the biologging meeting and the marine mammal biennial happening in September and October. We’ve also discussed loads of topics including statistical methods, funding awards and public outreach.

If you are an early careers researcher at the SOI you are very welcome to join us, our meetings aim to address whatever our members feel they currently need, providing a responsive support system with a relaxed, friendly environment. Please visit our new website here to find out more, see when our next meetings are and sign up to the mailing list, or you could come along to our welcome day event on Tuesday 3rd October (next week) to meet some of us and chat about the group and early career life.

A SOI early career network meeting for practising talks and sharing presentation ideas

I’ll certainly be practising the various presentations I need to give in the coming month at the ECN! I’ve already been to one conference this month, the wonderful meeting of the British Neuroendocrinology Society in Nottingham where I got to present my work on oxytocin and behaviour in seals. Next I’ll be talking to the public about any and all aspects of grey seal life on the Isle of May, during their annual seal weekend. This happens to celebrate the start of the grey seal breeding season, and afterwards the island is then closed to the public for the rest of the year to protect the breeding seals from disturbance.

Presenting my oxytocin work at the BNS 2017 conference in Nottingham

I’ll then be travelling to the University of Edinburgh to talk about my work on oxytocin and behaviour on the 10th October. I can’t wait to meet everyone at the Centre for Integrative Physiology and hear all about their amazing research on neuroendocrinology, I got to meet a few lab members at the BNS 2017 conference and their studies on modelling oxytocin dynamics are fascinating. Finally I’ll be heading out to Canada towards the end of October to present our PHATS work at the 22nd Biennial Conference on Marine Mammals. Phew, it’s going to be a crazy month! If you’d like to know more about any of my work, feel free to say hi at any of these events, or you can keep up with me on Twitter (@KJRscience).

EVENTS:

1st October – Isle of May seal weekend

10th October – 3pm talk at the Centre for Integrative Physiology, University of Edinburgh

22nd-27th October – 22nd Biennial Conference on Marine Mammals, Halifax, Canada

Hauled out grey seals in East Tarbet in the north part of the Isle of May, Scotland

 

New Publication – An explant approach to studying fat tissue function in wild animals

Adult male grey seal hauled out on a rocky seashore. Even in wild conditions, the PHATS team is bringing cell culture into the field!

Link to article: https://www.nature.com/articles/s41598-017-06037-x

Or read the summary here on this site.

Behaviour 2017’s fantastic closing dinner party, complete with a live band featuring 6 ukuleles!

I have now returned from an incredibly successful week at Behaviour 2017 (link), and what a spectacular conference it was! The sheer variety of science that people were talking about was incredible and inspiring, plus I got a great response to both my symposium talk on seal oxytocin and the poster I presented on aggression. I meet so many wonderful people, heard lots of interesting talks and I even managed to avoid getting roasted in the blazing Portuguese sun! I had never previously been to a behaviour conference of any kind, but this one has really encouraged me to keep an eye out for future ASAB meetings to present at. Huge thanks to the lovely people working as part of the SoHaPi research group for inviting me to speak at your symposium, I look forward to meeting up with you all in the future!

Taking the stage at Behaviour 2017 to talk about my work on oxytocin in wild seals

More good news was waiting for me when I arrived home from Portugal; our PHATS team leader, Dr Kimberley Bennett, let us know that the first paper the PHATS team have worked on was coming out at last! This paper details our work investigating whether an explant approach (basically blobs of many living cells) would work for culturing fat (or adipose) cells collected from wild animal species in field conditions. Additionally, we wanted to know whether we could manipulate the explants during culture to

100mg explants of adipose tissue weighed out and ready for transfer to culture plates for their 24 hour exposures to different treatments on the Isle of May, Scotland.

uncover the physiological consequences of changes in the nutrients or hormones the cells have access to. We found we could not only keep our cells alive once collected from wild seals on the coast of Scotland, but once transported back to the lab we could culture the cell explants for at least 24 hours. During this time we could expose the adipose cells to different treatments, such as high glucose concentrations in the cell culture media (the sugary, salty goo that cells are suspended in during culture to keep them alive) or difference hormone additions, such as hydrocortisone. We found significant differences in the metabolic profiles of adipose cells given different treatments, demonstrating this technique could be used to test the responses of wild animal tissue to a variety of substrates an individual may physiologically generate, or be exposed to.

Studying wildlife physiology is always challenging because collecting samples is tricky, typically giving small samples sizes in less than ideal conditions for complex labwork. However our work to bring cell culture techniques to the wild regions of Scotland shows that even difficult processes like cell culture, which require sterile conditions, aseptic technique and specialised equipment, are possible with thought and preparation beforehand. Studying cell function in wild animals is important as how different tissues function in response to different environmental challenges will impact on how individuals survive. Fat tissue is especially crucial for survival as it represents the energy stores animals have to rely on when conditions are tough, and also helps keep individuals warm in cold environments. By understanding how fat tissue functions, we can better understand why different species in changing environments can either adapt to meet new energetic challenges or be overwhelmed by them.

Even in muddy, windy or wet conditions, cell culture experiments can be possible if you are careful! (grey seal mothers and pups on the Isle of May, Scotland)

Speaking of ‘the wild regions of Scotland’, it’s that time of year when I start prepping all the field equipment for the PHATS team’s annual research trip to the Isle of May grey seal breeding colony, off the east coast of Scotland. Join us here for our fieldwork blog, bringing you all the adventures we have running a tissue culture lab on an island full of seals. We are scheduled to leave in late October, and will stay on the island studying the seals for about 2 months, heading home just in time for Christmas (hopefully!). I’ve also got two more conferences to attend before I go off into the field, one in September in Nottingham, UK with the British Society for Neuroendocrinology and one in October in Halifax, Canada with the Society for Marine Mammalogy. If you are going to either and want to say hello I look forward to meeting you there!

Weaned grey seal pups occupying the path down to Kirkhaven harbour on the Isle of May, Scotland

Attending Behaviour 2017 and other upcoming conferences

Hear about all the hormone, behaviour and adipose tissue function work I’ve done with seals at any of the three conferences I’m attending this year!

I’m going to Behaviour 2017 in Portugal next week!

Conference information: link

I’m going to be talking about my work on oxytocin and maternal and social behaviour in grey seals on monday afternoon, plus I’m presenting a poster on the development of aggressivness in seal pups on wednesday and thursday.

Symposium talk: Syposium 1 on Monday 31st July at 17:35 – 18:05

The symposium is titled ‘How Social Behaviour can impact individual health and fitness’. It will feature talks looking at how social living can impact on a range of aspects of an individual’s physiology, and the potential fitness costs and benefits associated with them. The talks cover primate species, fish and of course seals in my case!

Poster: Poster 278, Wed + Thurs, 2nd-3rd August 14:00 – 16:00

If you’re going to the conference and would like to find out more about my work it would be great to meet you there!

Please do say hello if you would like to talk to me about my research, my crazy ginger hair usually make me easy to find!

I’m also attending two other conferences this year, one to (hopefully) talk about my oxytocin work and the other to talk about the tissue culture work I’ve done

(TBC) Oxytocin work – 10th – 12th September
British Society for Neuroendocrinology, Nottingham (conference site: link)

Tissue Culture work – 22nd – 27th October
22nd Marine Mammal Biennial, Canada (conference site: link)
“An explant approach to understand adipose tissue function; metabolic profiles of blubber tissue differs between tissue depth, cell culture conditions and energetic state.”

So if you are attending either of these conferences you can catch me there too!

Safe travels!

New Publication – IV oxytocin causes pro-social behaviour in seals

Grey seals on the Isle of May, Scotland. Staying together is important for mother-infant pairs, especially on a dangerous seal colony.

Link to article: http://rspb.royalsocietypublishing.org/content/284/1855/20170554

Or read the summary here on this site.

This week has turned out to be a hectic but good one, I’ve returned from the University of Liege just in time for my next paper to be published in the Proceedings of the Royal Society B. The paper comes from the research in my NERC funded PhD with the Sea Mammal Research Unit, University of St Andrews on the hormone oxytocin and its impacts on social and maternal behaviour, rather than the pollutant research I’m currently doing with the PHATS team. Like much of my work, the study was done with weaned grey seal pups on the Isle of May, and involved giving the seals specially designed doses of oxytocin to see what (if any) social behaviours the hormone affected.

There have been lots of studies that show certain behaviours are linked to oxytocin concentrations (including some of my own grey seal work!), but the problem with correlations is that you have no idea which side of the relationship is driving things. For example, it would be impossible to tell using only correlations whether increased social behaviours are causing high oxytocin levels, or high oxytocin levels are triggering more social behaviours. Understanding causality in such hormone-behaviour relationships is important so you can identify the ‘cause’ and the ‘effect’ within the correlation. It can be difficult to do outside of laboratory settings however, as the only way to test for causality is to either increase the hormone’s concentration in an individual via manipulations or knock out the functionality of the hormone using antagonists. Due to these difficulties, there is only one study (apart from the one I published today) that has ever given oxytocin to wild individuals, and while they did find changes in pro-social behaviours they had no prior knowledge of the natural oxytocin-behaviour systems in their study animals.

We know high oxytocin grey seal mothers stay closer to their pups, but does the hormone cause the behaviour or does being near to their pup for more time cause greater oxytocin release?

In our study we were testing whether oxytocin triggers individuals to stay close to each other, as we know from grey seal mothers that the higher their oxytocin concentrations, the more time they spend close to their pups. We gave oxytocin and saline (control) treatments to weaned grey seals that had never previously met, and recorded their behaviours after the treatments. We found that oxytocin not only triggered individuals that had never met before to spend more time together, but also reduced aggression between the two and the amount the seals investigated each other, an indication of familiarity. This makes our study the first to verify a naturally existing oxytocin-behaviour relationship in wild individuals, which is very exciting. Studies like this have been done in captivity using domestic, laboratory or zoo animals but it’s crucial to study behaviour and physiology in natural settings with wild individuals, as no matter how hard you try you will never truly re-create all the complex aspects of wild environments in a captive setting.

Weaned grey seal pups associating on the Isle of May, Scotland

The treatments were all given intravenously (IV), as the more common, intranasal route of oxytocin manipulation was not possible with the weaned seal pups; they can close their nostrils and hold their breath for a long time! The success of this route of administering the manipulation means that other animal species, that may not be suitable for intranasal manipulations, could potentially have similar studies done on them in the future to help us understand more about oxytocin’s important role in bonding and behaviour. We also spent considerable effort designing the oxytocin dose given to the seals to be as low as possible while still having a behavioural affect. Many doses used in the scientific literature are much higher than natural concentrations, and there are concerns that generating high levels in study individuals could trigger behaviours that would never naturally happen, or have unexpected, and unwanted, side effects.

Weaned grey seal pups having a disagreement on the Isle of May. Reduction of aggression between familiar individuals happens naturally without oxytocin release in seals, but manipulations also trigger this behavioural change with seals that are complete strangers.

Despite the effort we went to in replicating natural oxytocin concentrations as much as possible for our study, the treatments still triggered some behaviours that are not naturally correlated to oxytocin release in seals. Low aggression and reduction of investigative behaviours are indications that seals recognise each other, and naturally occur after several days of living together, independently of oxytocin release. The behavioural changes in our study seals also unexpectedly persisted for several days, long after the dose would have been metabolised and broken down in the bloodstream. These unexpected effects show that we still have a lot to learn about oxytocin’s role in the formation and maintenance of social and parental bonds. If the hormone is going to be used to safely and successfully treat human psychological conditions such as schizophrenia, autism spectrum disorders and post traumatic stress disorder (and there have already been clinical oxytocin trials for such conditions in human subjects), then more research is needed into the powerful effects oxytocin can have on our behaviour and neurobiology.

Weaned grey seal pup on the Isle of May.

Liege 2017 – Goodbye to Liege and Conference plans

Lipids extracts from our seal blubber samples, ready for organochloride pesticide (OCP) analysis.

Just like that, my time at the University of Liege has finished and I’m back at the Sea Mammal Research Unit in Scotland. I was successful in preparing all our grey seal blubber samples for analysis, and now we just need to wait for the results from the Gas Chromatography – Mass Spectrometry (GC-MS)

The spectacular Liege Guillemins railway station, waiting for my ride home!

machines. We will hopefully get all our results by July, and in the meantime I will get back to the bichemical analysis of the samples from the tissue culture experiments on the Isle of May last year. My last few days in Liege flew by, a whirl of labwork, tasty Belgian fries and one last trip to Masion du Peket to enjoy their delicious drinks!

 

Weaned grey seal pups interacting on the Isle of May

We’re not just focusing on labwork here on the PHATS team however. We’ve been working hard on analysing our data and are now ready to start getting our science out there! We’ll hopefully be attending conferences this year to present our findings, and if you are interested in our work do come and find us at the below venues. I will also hopefully be presenting some of my work on the hormone oxytocin and it’s affects on bonding, social and maternal behaviour in seals. While the blog will be on hiatus until we return to the field in October, we will update it when we attend or present at conferences, or if we publish any papers on our work so watch this space!

Upcoming conferences:

30th July – 4th August: Behaviour 2017 (ASAB summer meeting & 35th International Ethological Conference)

22nd – 27th October: 22nd Biennial Conference on the Biology of Marine Mammals (Society for Marine Mammology)

Study seals Alpha, Kilo and Hotel on the Isle of May, 2016

 

Liege 2017 – PCBs, PBDEs and OCPs

Samples undergoing acid purification and agitation for OCP analysis

My last week at the University of Liege has arrived, and I’m working hard to ensure that all the PHATS team’s labwork is complete before I leave to return home. All of our samples are now ready for analysis that will let us detect PCB and PBDE levels in our Scottish grey seals. PCBs and PBDEs are two types of the many persistent organic pollutants (POPs) that are present in our environment. I am currently working on preparing our samples for another type of analysis that will enable us to detect a third kind,  OCPs. As POPs in our environment, and PCBs in particular, are still currently in the news after the recent revelation of just how highly contaminated with PCBs some marine mammals are becoming, I thought I’d spend this blog introducing the three types of POP I work on and why they are so problematic.

Samples after acid purification, showing the clear fraction I need to collect for OCP analysis

PCBs, or polychlorinated biphenyls, are pollutants that are made up of two linked rings of carbon atoms with a varying number of hydrogen and chlorine atoms bound to the rings at different positions. There are many possible combinations of the number and locations of the hydrogen and chlorine atoms binding to the rings, and these give rise to the large variety of PCBs (called congeners) that exist. Approximately 130 different types of PCB are found in commercial products, and they can be divided into two groups (dioxin-like and non-dioxin-like) based on their structure and toxicity.  PCB production was banned in the USA in 1979 and by the Stockholm convention (signed by over 150 countries worldwide) in 2001, however they persist in our environment due to their slow degradation rates. One of the main reasons PCBs were previously manufactured and used in industry was their inert properties; only incineration at high temperatures can safely destroy them. Previous uses of PCBs include in coolants and lubricating oils, paints and electric wire coatings.

Orca have some of the highest measured POP concentrations in an organism worldwide due to their high position in the food chain.

PBDEs, or Polybrominated diphenyl ethers, are also made up of two carbon rings, but they have bromine bound to the rings rather than chlorine. The fewer the bromine atoms per molecule of PBDE, the more dangerous they are considered to be as congeners with between 1-5 bromine atoms bioaccumulate more effectively in living organisms. PBDEs are still being manufactured and widely used in many man-made products, the Stockholme convention which banned PCBs only restricted the production of some PBDEs. Some states in the USA have begun prohibiting their manufacture and use in the last decade however. PBDEs are flame retardant and are therefore commonly incorporated into electronics, plastics, fabrics and other building materials.

Bald eagles severely declined in the mid 20th centuary until the ban on DDT use in the USA. Bioaccumulation of the pesticide up the food chain affected the formation of their eggs, leading to thin shells that broke under the weight of an adult incubating them.

OCPs, or organochlorine pesticides, contain carbon, hydrogen and at least one bound chlorine atom but do not contain carbon ring structures like PCBs and PBDEs. There are many different types of OCP, however arguably the most well known is DDT (Dichlorodiphenyltrichloroethane) which was heavily used as a pesticide across the world to kill insects for both agricultural and disease control purposes. The famous book ‘Silent Spring’, written by Rachel Carson in the 1960s, is all about OCPs and the negative impact overuse of pesticides has on the environment. The production and use of some OCPs like DDT and heptachlor has been strictly limited by the Stockholme convention. Due to their efficiency at killing insects, their use is still permitted in some circumstances, such as the use of DDT to control mosquitoes that carry diseases like malaria.

POPs have been connected to a wide range of negative health impacts in both people and wildlife, and chronic exposure to any type of POP will cause problems for any organism. All POPs are carcinogenic (cancer causing) and are potent endocrine disruptors, interfering with growth and development, immune function and reproductive systems. There is growing evidence that POPs impact on obesity, leading them to be labelled as ‘obesogens’. The PHATS project I am part of is hoping to uncover some of the underlying physiological and genetic mechanisms that influence fat tissue function and determine how POPs can interfere with these processes. By studying a marine mammal species which has lots of fat and lots of bioaccumulated POPs, we can gain a better understanding of how these chemicals have such far reaching and devastating impacts on our health and the environment.

One of the PHATS team study animals from the Isle of May 2016, ‘Mike’, a newly weaned grey seal pup. Even though she is only a month old, ‘Mike’ will likely have high concentrations of POPs in her tissue due to the high position in the food chain (trophic level) seals occupy in the UK and the fact that mothers pass a large proportion of their accumulated pollutants onto their infants via their milk.

Liege 2017 – PCBs in the news, the most contaminated whale in the world

Purification columns being prepared with hexane for the lipid extracts from our blubber samples to be added.

It’s been another busy week here in chemistry labs at the University of Liege. I’ve completed extracting all the PHATS team’s blubber samples for persistent organic pollutant (POP) analysis, and now am moving on to the purification part of the sample preparation process. I’ve only got two weeks left to get all the sample preparation completed, so hopefully all the lab work will go according to plan! The purification process isn’t too complicated but it does have lots of time consuming steps, from multiple standard spikes, to acid clean-up on columns, to concentrating the samples down via nitrogen evaporation. So it’s just a case of getting your head down and getting on with it all, as the sooner it’s done the sooner we’ll have some interesting results to look through.

Purification columns, with samples from Echo to Kilo undergoing acid clean up. You can see how ‘dirty’ the samples are from the brown/black sludge that builds up in the columns!

The results of POP studies are frequently worrying as well as interesting. A good example of this happened last week, when the Scottish Marine Animal Stranding Scheme (SMASS) got some lab results back showing the PCB concentrations in one of the stranded whales they had examined last year, ‘Lulu’, one of Scotland’s few resident orca. She sadly had one of the highest ever recorded concentrations of PCBs in her body, and there are concerns that the other members of her pod will have similarly high levels. Another interesting (and sad) aspect of Lulu’s case is that she had never produced a calf, despite the fact she was about 20 years old and orca usually have their first calves at around 14 years of age. It is well known that POPs negatively impact on individual health, including fertility, therefore it is possible Lulu failed to reproduce due to her high pollutant burden. Even more concerning however, is what might have happened to Lulu’s high PCB concentrations if she had produced a calf.

Orca pod with young calves. The females will unwittingly pass large proportions of their pollutant burden to their infants, meanwhile males will steadily accumulate POPs all their lives.

Female marine mammals pass approximately 60% of certain types of the pollutants they have accumulated in their blubber to their first calf, some passing through the placenta but the majority transferring via the fat rich milk marine mammals produce. Therefore, if Lulu had produced a calf, it also could have had one of the highest PCB burdens ever recorded in a marine mammal. Male marine mammals typically have much higher POP concentrations than adult females due to this phenomenon, although even male orca in populations considered to be ‘highly contaminated’ (251.2mg/kg) have far lower concentrations than Lulu did (957mg/kg). This sex pattern in pollutant concentrations is present throughout all marine mammals, and after first reproduction an adult female’s POP concentration will gradually decrease with each infant she produces. This means that infant marine mammals are typically exposed to dangerously high concentrations of POPs as soon as they are born. Interestingly, in the two marine mammal species that appear to show menopause (orca and short-finned pilot whale), upon reproductive senescence a female’s POP concentrations begin to increase once again.

Humpback whale eating sand eels off the coast of North America. POP concentrations in fish eating marine mammals are usually lower than those that eat other marine mammal species to survive, but are also effected by how industrialised the environment where they forage is.

Another major cause of patterns in POP concentrations in marine mammals is their position in the food chain (their trophic level) and the region they obtain their food from. Orca represent a fascinating opportunity to study these patterns as through-out the species, there are different populations that specialise in eating either fish or other marine mammals, or in other words different orca populations can occupy different tropic levels of a food chain. Groups that eat marine mammals, such as seals, sea lions and porpoises, typically have over double the concentrations of POPs in them than fish eating groups. This happens because the pollutants have become concentrated up the food chain due to bioaccumulation, where a predator eating lots of smaller prey gets all the pollutants in each individual it eats. A whale eating lots of seals to survive will accumulate all the pollutants all the seals were exposed to, and all the pollutants all the fish those seals ate too. Meanwhile a fish eating individual will ‘only’ accumulate the pollutants from the fish it eats. Additionally, individuals that hunt in highly industrialised areas have higher concentrations than those in ‘pristine’ areas, because the more POPs that are in a local area, the higher the concentrations in all the organisms from the bottom of the food chain to the top.

Studying patterns of POP concentrations in different types of individuals can therefore lead to a better understanding of how these persistent pollutants ‘move’ through organisms and can be transferred into later generations. It is not hard to see why POPs continue to be a problem for animal and human health, despite being banned decades ago.

Liege 2017 – A brief introduction to blubber tissue

Minced blubber biopsies in cells ready to be capped and put through accelerated solvent extraction, to obtain all the lipids from a samples for further analysis.
Weighing a ASE vial after ASE is finished and the solvent has been evapourated off to calculate lipid mass in the sample. This is from one of Foxtrot’s biopsies from last year, and it had 0.42g of lipid in it, meaning 80% of the original biopsy was fat.

Well my first week at the University of Liege working with CART has flown by, and I’ve been working on the blubber biopsies we collected from the grey seals last year on the Isle of May. All the lipids (fats) need to be extracted from the blubber tissue before we can move forward with the pollutant analysis, so all the samples must be carefully prepared and put through Accelerated Solvent Extraction (ASE). This process uses high pressure and temperature conditions plus chemicals called solvents (like hexane and acetone) to remove all the lipids from the sample in the cells. This process gives us a completely liquid solution of lipids and solvents at the end of it, and we can then evaporate the solvent to leave just the lipids from our sample. This step is important as it gives the lipid mass of our sample, and allows us to work out how many nanograms of pollutant per gram of lipid in our sample there is (ng/g) . While ASE of our samples is an important step in the lab work, there isn’t really much more to say about it so I’m going to use the rest of the blog this week to give a brief introduction to blubber tissue, a crucial part of the anatomy of all marine mammal species worldwide.

Blubber tissue enables marine mammals to endure cold aquatic environments, like this Orca family living off the coast of Iceland.

All marine mammals, from the largest whale to the smallest seal, have a layer of fat underneath their skin called blubber. This layer of fat is extremely important for the survival of marine mammals for two reasons:

  1. It enables them to keep warm (thermoregulate) in freezing oceans.
  2. It provides a store of energy for individuals to utilise when they are not feeding, which happens in many marine mammal species at various points throughout their lives due to breeding or moulting.
A blubber biopsy from one of our seals. Blubber samples for pollutant analysis must be stored in glass or wrapped in foil to prevent loss of pollutants from the sample to any plastic they come into contact with.

Fat tissue deposits in all animal species perform these same two functions, however other species frequently have additional ways to thermoregulate (such as fur in land mammals) or do not endure long periods of fasting repeatedly while migrating or breeding as many marine mammals do. The importance of this tissue has lead to substantial blubber thickness evolving in marine mammals, and a stratified structure throughout the depth of the tissue is present so that it can perform both functions at the same time. Typically, blubber tissue can be roughly divided into three sections as you go from the part closest to the skin (the outer blubber) to the part closest to the inside of the seal (the inner blubber). The inner blubber is the most metabolically active, and this is where lipids are mobilised to provide energy for an individual when it either cannot find food or is purposefully fasting. The mid blubber is the most variable in thickness across individual marine mammals, and in thin individuals can be completely absent. It is thought it acts as a more long term storage tissue, and that its thickness is influenced by seasonal food availability. The outer blubber is typically of stable thickness within a species regardless of the nutritional state of an individual, and is thought to be primarily for thermoregulation. Hence even starving individuals will always have some blubber tissue to keep them from freezing, as the outer blubber is not mobilised as an energetic resource.

Blubber is a fascinating tissue to study and several different approaches can be used to analyse it in many contexts, like this recent study by one of my friends, Joanna Kershaw, who measured the hormone cortisol in blubber from porpoises to validate it’s use as a biomarker of body condition. The PHATS project I work on uses both established techniques (investigating pollutant concentrations) and novel protocols (the explant approach for tissue culture experiments that our team leader pioneered in seals) to make the most of the blubber samples we collect from our study animals to explore the prevalence of persistent organic pollutants in the marine environment and it’s impact on energy balance in seals.

A particulary tubby looking Charlie from last year’s study group on the Isle of May. Her fat reserves in her blubber layer will help her survive the tough first year at sea she faces when leaving the breeding colony.

MEANWHILE I am settling back into Liege life quite happily outside of the lab. I am not staying in the university accomodation this year, and have a lovely little flat not far from the campus to retreat to. In my time away from the lab I’m trying to keep up with the usual paper and grant writting that all resarchers need to keep on top of, plus greatly enjoying bebing reunited with the amazing macaroons they make here! Seriously, why can’t they be this good in Scotland…

Happiness is macaroons =D

PHATS project progress, ELISA validations and my return to Liege

Moulting grey seals hauled out on the Isle of May
Remember please send all ringed shag sightings to shags@ceh.ac.uk

Welcome back to my corner of the internet and the PHATS blog! The first four months of 2017 have flown by as the team has headed back into their labs to analyse all the samples we collected over the winter on the Isle of May grey seal breeding colony (to read about our fieldwork adventures, see these blogs here). I was lucky enough to escape to perform a survey of the Isle of May in January, to see if there were any grey seals that were moulting early in the year. There were plenty of them as it turned out, which bodes well for the fieldwork we are planning next year to try and look at moulting seal physiology. The island already looks so different to how it was when the seals breed there in the winter, much greener and all the seabirds are starting to come back. The cliffs were lined with guillemots, razorbills and shags; some were even getting started on gathering nesting material. The puffins had not returned yet, they arrive later in the year closer to summer, but we did see a few on the water during the boat crossing to the island.

Large grey seal haul out on the North East coast of the Isle of May
Is there anything better than well organised samples?

Inside the lab, I’ve been working on biochemical analysis of the cell culture media from all the blubber sample experiments last year (see here for more info) and am now two thirds through the samples we generated. By measuring the metabolic profiles of the various blubber culture experiments, we can see if the pollutant or hormone treatments had any impact on the blubber cells we collected from the seals. I’m also working on validating ELISAs (Enzyme-linked immunosorbent assays) to detect a variety of hormones in the blood samples we collected from the seals last year, so we can see if an individual’s hormone profiles are linked to their pollutant burden.

Using ELISAs on wild animal species like grey seals can be tricky, as they use antibodies as part of a binding process to detect the hormone you are interested in. These antibodies will have come from a specific species of mammal, usually rodent or domestic animal species that lots of scientists study, and the company making the ELISA kit will provide a list of species they know the kit works with. As a hormone’s protein structure is not always the same in different species of animals, the antibodies used in a widely available ELISA kit may not react properly with samples from unusual species that has never been tested with that kit before. Unfortunately seals often fall into the ‘unusual’ category, so we need to test the kits (validate them) before we use them to run lots of our samples, to make sure the results we are getting from the kits are accurate. There are several things to check when validating an ELISA, some of the most important are:

  1. Test for linearity. By diluting some of your samples (e.g. to half the concentration, then a quarter, then an eighth etc) you can make a serial dilution series to run on the kit. You can then see whether the curve the dilution series produces is parallel to the standard curve, which is what the kit uses to determine hormone concentrations. If the curve is not parallel to the standard curve, then the hormone in your samples is not binding correctly to the kit components.
  2. Test for recovery. By spiking a sample with a known quantity of the hormone you are interested in studying, you can tell how much the kit is detecting and how much is ‘lost’ during the analysis process.
  3. Test for consistency across kits and within kits. Many studies have lots of samples and need to use more than one kit to analyse them all. You must make sure the results of one kit are comparable to the others (inter-assay coefficient of variance), and the easiest way to do this is to run the same sample on each kit you use. You can then calculate the coefficient of variance across all the kits you have run. It’s also important to check the kit’s internal consistency (intra-assay coefficient of variance) by running one sample multiple times on a plate and seeing how similar the results are, and by running all samples in duplicate on the kit.

Our seal blood samples are proving to be rather tricky currently, and we’re still working on which are the best kits to use to measure the hormones we are interested in. The biochemistry analysis is going very well though, and we’re all looking forward to having some data to play with in the coming months.

Two ELISA plates at different stages of incubation. The different intensities of colour indicate different concentrations of hormone.

The biochemistry and ELISA work I’m doing is currently on hold however, as I have returned to the University of Liege in Belgium to work with the Centre de Recherche Analytique et Technologique (CART) to detect the amounts of persistent organic pollutants (POPs) in the blubber of our study seals from the Isle of May last year. We will be using Gas Chromatography – Mass Spectrometry (GC-MS) again, which requires a lengthy extraction and clean up process before the blubber tissue can be analysed (see these blogs from last year for more details) so I will be here for a month to work on our samples (and I will update the blog every week while I am visiting CART). It is always interesting, and more than a little sad, to find out how many pollutants all the seals have inside them after we got to know so well during the field season…

Unfortunately Alpha and Kilo, like all young marine mammals, will have large concentrations of POPs in their tissue from the high fat milk they drink from their mothers.