Biomarkers Flashcards
What three ‘biomarkers’ are looked at?
- Stable Isotopes
- food webs
- diet shifts
- uptake experiments - Fatty acids
- food webs and dietary source markers
- diet shifts - Algal pigments
- dietary selectivity
Recap - how are stable isotopes used
- By far the most common application of SIA in trophodynamic research is the derivation of non-integer trophic level/position
- (a tool to infer trophic position)
- The utility of stable isotope techniques depends primarily on differences in the isotopic ratio (e.g. 15N:14N or 13C:12C) among consumers and their diet
- These differences occur due to preferential retention of heavier isotopes and excretion of lighter isotopes during assimilation, metabolism and excretion
- This change is referred to as trophic fractionation (Δ)
What is a diet switch experiment?
- Changing a study organsims diet and seeing how long its carbon and nitrogen levels take to reach the new equilibrium - a fundamental component of most food webs.
- What is the step up or down in carbon and nitrogen ewhen a diet is changed?
Crassostrea gigas and Mytilus edulis – diet switch experiments
Carbon and Nitrogen fractionation calculated as difference between food source and consumers after equilibration
Δδ13C – 1.85; 2.17 Δδ15N – 3.79; 3.78
Dubois et al., (2007) J. Exp. Mar. Biol. Ecol. 351: 121-128
What assumptions are the absolute values of trophic level based on?
- Absolute values of trophic level are dependent on assumptions about isotopic signatures at the base of the food chain
- d15Nbase for example is positively correlated with seabed temperature and salinity (Jennings et al., 2003, Mar. Biol. 142: 1131-1140) while d13Cbase is driven by seabed temperature alone (Barnes et al., 2009, Est., Coast. Shelf Sci. 81: 368-374)
- These variables are pronounced in coastal areas making spatial comparisons of trophic level easily biased if fine-scale information on base δ15N or δ13C is not available
- These differences in δHXbase are utilised to great effect in large scale migration studies (Trueman et al., 2012, J. Fish Biol. 81(2): 826-847)
Relative differences in mean isotopic signature among species are used to infer trophic structure
Conventionally represented as a δ13C-δ15N bi-plot
What aspects of the food web do they provide an insight into?
Bi-plots are semi-qualitative and provide valuable insight into many aspects of food web structure e.g.
- temporal changes in food web structure as a result of species invasion (Vander Zanden et al., 1999, Nature 401: 464-467)
- spatial variation in the extent to which terrestrial carbon supports coastal production (Darnaude et al., 2004, Oecologia 138: 325-332)
Letessier et al. 2012 J. Mar. Syst. 94: 174-184 – Zooplankton of the MAR
Example stable isotope study
- Had two sights which sat north and south over the big fracture system over the North Atlantic ridge
- δ15N values spanned >range in the north than in the south
- Much tighter clustering of what’s going on the south even though there are similar species
- Within-species values differed by location
- species feeding at different trophic levels depending on whether they are north or south of this frontal system.
- Significant differences in δ13C north and south of sub-polar front
- Themisto compressa (amphipod) shows broad dietary variability (3.7%o) difference in δ15N
- Euphausids E. krohni and N. megalops had similar ranges – but ones is 15N enriched compared to other suggesting reduced competition between sympatric species
- another example looking at shorter food chains living of abyssal muds
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Reid et al., 2012, Mar. Ecol. Prog. Ser. 463: 89-103 – MAR benthic food web
Drawing together different parts of stable isotope analysis.
This is work I did with Will on the Mid Atlantic Ridge, looking at the entire community, again still N and S of the fracture zone. Looking at different temporal patterns to see if the food web is retained in its structure from year to year - whether you see seasonal variability.
- Analysis of δ34S indicated no input from chemosynthetic sources
- Large discrimination in δ13C between consumers and food source
- Differences between trophic guilds (SDF, SSDF, SF, P/S) - how these animals are functioning
- 2 dominant trophic pathways - strong correlations between δ15N and δ13C
Mid Atlantic Ridge conceptual food-web model
What ways can food arrive in the deep sea?
- Traditonal - phytoplankton blooms - can get intercepted by filter feeders, then the signature will pass through the deposit feeders (green pathway)
- The other way is through a conveyor belt of trophic recycling through the pelagic system.
- Blue pathway - conveyor of different trophic recycling within the pelagic system leading down to food falls.
- The big problem in any of these systems is working out what the bacteria are doing
- Are some of these surface deposit feeders simply consuming phytodetritus because it acts as an incubation base for bacteria in their gut biome- and its actually bacteria that are producing the nutrients
Reid et al., (2013) Deep-Sea Res. II 98: 412-420 – spatial and temporal size-based trophodynamics of MAR fishes
Carbon -
Antimora rostrata
- blue hake
For all the stations and time periods, that the carbon signature is becoming more enriched with time. Moving from being slightly piscivorous to being larger scavengers.
- δ13C increase with size
- trend not varying spatially
Coryphaenoides armatus – big scavenging rat tail – you do see spatial variability trends. Strong diet switch at the southern site.
- δ13C increase and decrease with size
- trend varies temporally 2007 v 2009
Reid et al., (2013) Deep-Sea Res. II 98: 412-420 – spatial and temporal size-based trophodynamics of MAR fishes
Nitrogen - to see if they are feeding higher up the food chain
Antimora rostrata -
- δ15N increase with size
- As they get bigger, they can feed on bigger things - important to remember that this does not mean that they stop feeding on smaller things
- trend not varying spatially or temporally
Coryphaenoides brevibarbis
- δ15N increase with size
- slope varies spatially
- different feeding patterns?
- Variability in δ15N and δ13C explained by body size in all species
- Trends in A. rostrata and H. macrochir indicate an ontogenetic increase in TP and they are feeding higher up in that food web
- Difficult for other species; potential shifts in the resource they are utilising? TP increases?
- Indicates varying degree of overlap in trophic ecology and feeding plasticity
What paper merges fatty acid profiles and stable isotopes?
Stevens et al., (2008) Mar. Ecol. Prog. Ser. 356: 225-237 – ontogenetic diet shifts in vent shrimp
- Juvenile Opaepele loihi (pacific vent shrimp) have variable isotopic compositions related to size
- as they grow they are shifting their carbon and nitrogen and reaching some sort of stability in the larger sizes
- >25mm enriched in 13C and 15N cf. ≤25mm
- Bacterial mats and detritus were more depleted in 13C
- Implying juveniles are more associated with a bacterial mat, and as they grow they are switching to something else.
- Supplemented by fatty acid analysis – ontogenetic changes in neutral lipid composition
- As the animals are growing you are getting an increase in the monounsaturated fatty acids and a decrease in the polyunsaturated fatty acids
- ≤25mm PUFA = 21-65% total lipid
- As the animals are growing you are getting an increase in the monounsaturated fatty acids and a decrease in the polyunsaturated fatty acids
- data implies a diet switch: bacterivore to scavenger
What are fatty acids? (don’t need to know exactly)
- FAs consist of a terminal methyl group (CH3), a carbon chain, and a terminal carboxyl group (COOH)
- Most naturally occurring FAs have an even number of carbons (14-24)
- Saturated FAs (SFAs) have no double bonds
- Monounsaturated FAs (MUFAs) have 1 double bond
- Polyunsaturated FAs (PUFAs) have 2-6 double bonds (typically)
- Separated by a methylene group (CH2)
Notation – A:B(n-x)
A = number of carbons
B = number of double bonds
X = position of a first double bond relative to methyl group
DHA = 22:6(n-3)
What can fatty acids be used to do?
- FAs have been used extensively in pelagic food webs to identify phytoplankton and characterise the diets of zooplankton (Dalsgaard et al., 2003, Adv. Mar. Biol. 46: 225-340)
- Diatoms and dinoflagellates have distinctive FA compositions and zooplankton retain dietary FAs in the tissues; Calanoid copepods also synthesise a few rare FAs useful to assess their role in diets of higher consumers
- Top predators – dietary FAs deposited in the blubber of marine mammals with little modification allowing good characterisation of diet (Budge et al., 2006, Mar. Mamm. Sci. 22: 759-801)
- Unlike pelagic systems, benthic food webs are supported by a diversity of primary producers – although they have characteristic FAs a large number of potential food sources can pose problems in distinguishing their contributions to the diet
Useful signatures
Algae
(don’t need to know in great detail)
Algae – synthesis occurs in chloroplasts results in production of SFAs (16:0) (16 carbons) via acetyl-CoA
- Modified by elongation and desaturation to produce various unsaturated FAs
- Conversion from 18:1(n-9) to 18:2(n-6) requires desaturase enzymes found only in primary producers
- These 2 PUFAs are called ‘essential’ FAs as animals must obtain them from their diet rather than modifying other FAs
Useful signatures
Marine Bacteria
(don’t need to know in great detail
Marine bacteria – play an important role as a food source for deposit feeders and suspension feeders and as endosymbionts
- •Cyanobacteria dominated by 16:0 and 14:0
- •FA biosynthesis in heterotrophic bacteria produces odd-numbered and branched FAs such as 15:0, 17:0, 15:1 and 17:1
- Odd-numbered fatty acids come from bacteria
- •16:1(n-7) and 18:1(n-7) are typically dominant and useful tracers of bacterial contribution to diet
- •PUFA synthesis is rare: cold-water and deep-sea exceptions
Drazen et al., (2008) Comp. Bioch. Physiol. B 151: 79-87 – FA composition of abyssal holothurians and ophiuroids from NE Pacific
Explain this graph.
First one looking at bulk compositional differences. The first one is looking at wax esters and the glycerides are your general storage lipids. Lots of storage lipids is implying that the animal is feeding effectively and actually putting something away. Phospholipids are the component that it is getting from the diet, and forms and important function.
The amount of lipids different organism store related to a variety of physiological and environmental factors. Ophiuroidea tends to store more than the sea cucumbers, probably to do with a less continuous mode of feeding.
This is reflected as well within our phospholipids, looking at the different groups. Mono/poly/un saturated fatty acids and fatty acids indicative of bacterial origin. Interestingly can see quite a significant contribution of bacterial fatty acids to all of these groups. The fatty acid profile overall shows a high concentration of phospholipids. (lots of PUFA).
PUFA is a good place to look for differences in dietary requirements.
Look fatty acid profiles are particularly important to look for patterns in?
- Odd numbers – bacterial origin.
- 18:1n-7 is an important indicator of bacterial synthesis of phytoplankton detritus.
- Essential fatty acids that can’t be synthesized EPA – important for maintaining membrane fluidity.
- DPA and DHA.
- THA
Study for looking at the importance of bacterial input of food?
Howell et al., 2003, Mar. Ecol. Prog. Ser. 255: 193-206 – feeding ecology of deep-sea seastars
North Atlantic sea stars
- Analysis of 9 species – across 1,053-4,840m depth range
- PUFAs the most abundant FA in total lipids of all species – dominated by 20:5(n-3) and 20:4(n-6)
- as we saw in the previous study
- MUFAs; 20:1(n-13)(n-9) also abundant and non-methylene interrupted dienes (NMIDs) present in relatively high levels
- NMIDS are really indicative of bacterially derived material ingesting into their diet
- What fatty acid signatures tell us about these organisms maps quite nicely onto their functional role.
- Three identifiable feeding guilds (trophic groups)
- SF showed greatest reliance on photosynthetic carbon – high abundance of FAs characteristic of microplankton
- Mud ingesters relied heavily on heterotrophic bacterial carbon – high 18:1(n-7) and NMIDs
Fatty acid - study showing seasonal variability
Hudson et al., (2004) Mar. Ecol. Prog. Ser. 281: 109-120 – temporal variability in deep-sea holothurians
- Abyssal Amperima rosea and bathyal Bathyplotes natans show seasonal variation in FA composition
- the animals in March are in the most starved state as its the longest till their next food pulse
- PUFAs dominated in muscle tissue (post-bloom)
- meaning its a dietary derived signature
- 20:4(n-3), 20:5(n-3) EPA and 22:6(n-3) DHA
- Common characteristic fatty acids which are of important - essential fatty acids that they need to maintain membrane fluidity and then to get higher levels from their diet.
- Figure shows how these groups will separate out based on the composition and abundance of the fatty acids in their tissues.
- Pre-bloom; lower PUFAs, elevated MUFAs especially 18:1(n-7) and also elevated NMIDs - over winter when food availability is poor you are seeing a higher reliance on bacterially derived fatty acids in their nutrition.
- not the same for every species
Pond et al., (2000) Mar. Ecol. Prog. Ser. 198: 171-179 – unusual composition of storage lipids in vent shrimp (Rimicaris exoculata)
The interesting thing is that juveniles are packed full of these wax esters storage lipids compared to the larger size classes.
- Wax esters and triacylglycerols contained large amounts of PUFA (89%)
- Juvenile shrimp lipids - wax esters (56%)
- Bacterial/detrial material collected from vent sites contained very low PUFA (1%)
- Juveniles are not getting their food from that source
- FAs of juvenile shrimp dominated by 22:6(n-3)
- key DHA derived phytoplankton signature
- Wax ester more enriched than polar lipid
- Adult shrimp contained much less (n-3) FAs in their polar and TAG (storage) lipids
- Adult shrimp also contained more 18:2(n-4) ; 18:2(n-7) and NMIDs (bacterial signatures)
- Specialised lipid metabolism to store PUFA during early planktonic life stages
- Show juveniles feed more in the water column and come back to the vent as they grow.
What are algal pigments?
- Phytopigments, which include carotenoids, chlorophyll and the degradation products of chlorophyll have been used to indicate the ‘freshness’ of sedimenting organic matter
- As phytoplankton taxa have unique pigment signatures they can be used to track bloom composition and subsequent export flux
- Carotenoids can only be synthesised de novo by plants and some bacteria and are diagnostic of specific algal groups;
- 19’hexanoyloxyfucoxanthin – common pigment you find in prymnesiophytes and some dinoflagellates
- Violaxanthin - eustimatophytes (pico-nano phytoplankton)
- Diadinoxanthin - diatoms
Why are algal pigments important?
Algal pigment tell us about partitioning resource, and have an important role to plan, especially in reproduction. May explain population shifts, and why some animals are being successful over others, maybe its related the availability, and ability of these organisms to incorporate some of these compounds. Larval development s greatly improved if you increase beta carotene levels.
This paper looked at pigments in the guts and tissues of deposit feeding sea cucumbers from the abyssal North Atlantic.
To look at variability and separation of the marine snow.
Can see the main different groups, dark bars from the phytodetritus and the light bars are what you find in the sediment.
The phytodetritus sitting on top of the sediment is really enriched compared to the mud itself, therefore surface deposit feeders are going to gain much more access to this resource pool.
All higher in 2004 v 2005 - seasonal variation
All species had >[Chl a] in guts than sediment/phytodetritus with exception of M. Blakei - so they are assimilating that
Ovarian [carotenoid] varied considerably among species
[Chl a] 2004>2005 for O. mutabilis and P. longicauda
- Supports the observations that there is not a consistent food supply to deep-sea
- Temporal variability in ‘freshness’ - high flux events linked to fresher OM
Summary
- Biomarkers can elucidate physiological and biochemical pathways – especially over spatial and temporal scales (and habitats) where other techniques would prove prohibitive
- Stable isotopes have been used extensively to investigate food webs and trophic relationship
- Also used to investigate migration, assimilation and uptake rates – compound specific isotopic techniques provide greater resolution and used in conjunction with fatty acid analysis they can resolve the importance of individual dietary components
- Fatty acid analysis is not just for dietary stuides
- Algal pigments can identofy composition of detrital food and selectivity of detritivores (deposit feeders)
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