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