OCS Conference – Royal on the Park, Brisbane 19th-20th September 2013
Lipids, fatty acids and chondrichthyan ecophysiology. Heidi Pethybridge CSIRO Marine and Atmospheric Research, Hobart, Australia (
[email protected])
Abstract. Lipid and fatty acid analysis can provide a large amount of information on the condition, feeding ecology, and maturation status of marine organisms. The method has particular utility in the deep-sea and/or protected species, for example gulper sharks, where it is not practical or legal to collect large sample sizes. Here, we will use several recent examples for white shark, and species of dogfish shark, catshark, and chimaera, to illustrate how lipid-derived data, along with multivariate analyses, have be used to elucidate the diet, habitat-usage, condition, and life-history characteristics of individuals, populations and assemblages of chondrichthyans. This includes the assessment of maternal lipid investment during oocyte maturation and early embryonic development by viviparous and oviparous species. The current limitations of lipid and fatty acid approaches, and projected areas for future work, including the use of bioenergetics models, will also be discussed.
Lipids, fatty acids & shark ecophysiology: present uses and future directions Heidi Pethybridge, Peter Nichols, Ross Daley & Beth Fulton
OCS Conference, Brisbane 19-20th Sept 2013
Sharks occupy an important ecological niche as top-predators
Understanding shark trophic ecology is thus necessary to gain insight into their role in influencing niche, community and food web structure, and ecosystem dynamics. 2 | Lipids & shark ecophysiology| Research needs
They are also under increasing threat.......
Their disappearance could lead to an unbalanced ecosystem
3 | Lipids & shark ecophysiology| Research needs
But it’s not easy studying the ecology of sharks, particularly deep-sea dwellers and those that need protecting….
• Logistically difficult and costly – Expensive ship time – Expensive devices (submarines, cameras, CTD tags) – Limited resources (ships, people & equipment)
• Conventional methods time-consuming – Inherent biases associated with: • Stomach content analyses • Morphometric measurements – Large samples sizes required – Deceased specimens required (conservation concerns)
More robust , integrated and non-lethal techniques required..... 4 | Lipids & shark ecophysiology| Research needs
Biochemical tracers: Food-web, taxonomic & stock discrimination • Differentiate between what is assimilated and what is simply ingested : carbohydrates, proteins and lipids (and contaminants). – – – –
Stable or radio-isotopes : trophic position, niche areas, diet overlap Trace metals: diet, ecosystem health indices Genetics: prey composition, connectivity Hormones: reproductive status
– Lipids and their components: Lipids provide the densest form of energy, 37KJ/g Proteins and carbohydrates = 19KJ/g Many fatty acids are essential to early-life development, brain functioning, homeostasis, structural integrity, immune responsiveness, etc. 5 | Lipids & shark ecophysiology| Research needs
“You are what you eat”
What are Lipids? Lipid classes:
Fatty Acids: e.g. 18:0
Saturated fatty acid (SAT) Polar Lipids (PL)
Sterols (ST)
18:0/22:6
Free fatty acids (FFA) 22:6
Triacylglycerols (TAG) 18:0/22:6/20:5
Structural components of cell membranes
energy storage oxidation/ membrane stress stability/ hormone precursor
Monounsaturated fatty acid (MUFA)
e.g. 18:2w6
Polyunsaturated fatty acid (PUFA)
Lipids: Some examples of their applications…………. 0 • Total lipid 0 • Lipid classes • Fatty acids
condition indices energy allocation patterns trophic relationships
Lipids are also solvent & absorption carriers for organic contaminants . Thus, lipids are drivers of pollutant bioaccumulation.
7 | Lipids & shark ecophysiology| Lipids: a range of applications
Total lipid content: (1) Reflects an organisms physiological and nutritional condition (fitness).
In dogfish sharks: •Limited lipid content in muscle ( more coastal signature.
14 | Lipids & shark ecophysiology| Fatty acid profiles: trophic relationships among and within species
Fatty acid profiles: (3) Intra-specific ontogenetic and regional differences in diet of Greenland sharks (McMeans et al. 2012 JEMBE)
Fatty acid markers of: copepods
• decreased reliance on copepods with age. • increased dependency of seals.
Norway (KF)
seals
Canada (CS
• heavy reliance on pelagic teleosts and seals. Total length (cm) 15 | Lipids & shark ecophysiology| Fatty acid profiles: trophic relationships among and within species
Fatty acid profiles: (4) Maternal resource dependency in young bull sharks (Belicka et al. 2013 MEPS)
•20:3ω9 accumulate during periods of essential fatty acid (eg. 20:4ω6) deficiency. • High levels of the 20:3ω9 in sharks < 100 cm and suggest inadequate or undeveloped foraging skills by sharks and possible reliance on maternal provisioning. • Low levels in sharks >100 cm indicated no signs of essential fatty acid deficiency, implying adequate foraging.
16 | Lipids & shark ecophysiology| Fatty acid profiles: trophic relationships among and within species
Fatty acid profiles: (5) Validated approach based on a controlled feeding study on Port Jackson sharks (Beckmann et al. 2013 JEMBE) • Dietary FA are preferentially utilised by muscle after dietary change (within 3 weeks) • Muscle shorter term dietary changes, liver slightly longer
Muscle
Liver
Prawn
Squid
17 | Lipids & shark ecophysiology| Fatty acid profiles: trophic relationships among and within species
Cost: ≈$20-50 / sample Special materials: cryotubes or aluminum; NO plastics! Tissue/quantity: 0.5-1g of muscle, liver and/or gonad Total lipid content
extract oil
Lipid classes
Tissue dissections
Extraction
Methylation liberate Fatty acids Iatroscan (TLC-FID) GC & GC-MS
identify fatty acids
separate fatty acids
Chromatogram 18 | Lipids & shark ecophysiology| Lipid analyses – costs, materials and procedures
Recent technological advances: Fatty acid transmethylation (Parrish & Nichols In prep) Enhanced sample through-put to 400 fatty acid samples/week ! The trade-off is lipid class composition and total lipid data Total lipid content ≈ total fatty acids
Tissue dissections
Transmethylation
liberate Fatty acids
Lipid classes ST, WE
GC & GC-MS identify fatty acids
separate fatty acids
Chromatogram 19 | Lipids & shark ecophysiology| Lipid analyses – costs, materials and procedures
- Compared with profiles of known
Shark fatty acid profile
& potential prey species
(up to 60 variables)
SAT MUFA
PUFA
16:0 18:0 18:17 20:19 22:111 20:53 22:63 etc...
- Perform MDS analysis to identify groupings
Signature FA:
Myctophids:
copepods:
Squid:
Decapods:
18:19
22:19 20:111
22:56 18:19
20:53 (EPA) 16:0
16:0
20 | Lipids & shark ecophysiology| Multivariate statistical analyses
Amphipods: 20:53 (EPA) 22:63 (DHA)
Future work: (1) Refining and standardising multivariate data analyses
Data treatments • • • • • •
Transformations: normalise, log10, or non-transformed ? Percentage vs concentration data? Which fatty acids to include (up to 60 variables)? Which fatty acids to focus on: major vs minor? Biomarkers (ratios or sums of characteristic fatty acids) PerMANOVA – a new PRIMER tool
Discussions to be had at Ocean Science meet (Feb 2014) …..
21 | Lipids & shark ecophysiology| Future work
Develop bioenergetics, individual-based models based on Dynamic Energy Budget (DEB) theory (Kooijman 2000)
EVIRONMENT Food & Temperature
Future work: (2)
feces
Ingestion
Assimilation, pA
Reserve, E (J)
(0.7) Somatic Growth, pG
1- Maturation, pR
pM
pJ
Maintenance
• Conceptual and quantitative framework. • Describes energy and substrate (e.g. lipid) fluxes (J d-1) over entire life-cycle (egg to adult).
Catabolized energy, pC
Maturation, ER (J)= 0
Structure, V (cm3)
Larvae & Juveniles (
