Nicholas E.C. Fleming, Queen’s University, Belfast

Hydrothermal vent communities: food webs, trophic ecology and reservations about preservations Nicholas E.C. Fleming, Queen’s University, Belfast

“In the beginning” • Support for an alternative production pathway established using Stable Isotope Analysis (SIA) by Rau & Hedges (1979), Science. • This study used Bathymodiolusthermophilus • Marine phytoplankton typically between δ13C = -15‰ to -21‰ • Bathymodiolusthermophilus ca. δ13C = -33‰ • Deep sea vent ecosystems have very different elemental fluxes from shallow water and terrestrial counterparts

Stable isotopes in food webs: you are what you eat + x: Where x = Δ13C  1 ‰ and Δ15N  3.4 ‰.

SIA at HTV’s: the story so far.....

Problems with sampling protocols • A selection from the literature highlights: • Pre-analytical protocols differed between studies • No mitigation against preservation effects • Potentially the studies are hard to compare • Problem! • There are no standard protocols to harmonise future studies

Preservation effects Freezing δ15N δ15N δ15N δ15N δ15N δ15N Corbiculafluminea(Asiatic clam) δ13C δ13C δ15N δ15N δ15N δ13C Formalin Octopus vulgaris (Octopus) Ethanol Gadusmorhua(Cod) After Kaehler & Pakhomov (2001); Sweeting et al. (2004) ; Syvarantaet al. (2011);

Identifying preservation effects: Jellyfish Step 1. Does diet shift with increasing body mass? Fresh moon jellyfish Aurelia aurita show shift in δ15N of ca. 3.3 ‰ along size gradient Equivalent to 1 trophic level (mean trophic fractionation between consumer & prey = 3.4‰) Ontogenetic trophic shift Not all jellyfish are equal (e.g. in models of energy flow) This needs to be established for vent fauna Fleming et al. (2011) Marine Biology

Step 2. Effect of preservation on isotope values (Mean ± 95% confidence ellipse) • Identifying preservation effects: Jellyfish 2 Marked preservation effect e.g. in mean ± SD δ15N values: Fresh = 8.3 ± 0.9 ‰ Frozen = 10.3 ± 1.3 ‰ Alcohol = 10.7 ± 0.6 ‰ Preservation results in: - enriched δ15N values - overestimation of trophic position Is this the same for vent fauna? N = 45 Fleming et al. (2011) Marine Biology

Identifying preservation effects: Jellyfish 3 • Step 3. Effect of preservation on isotopic variation with size δ15N vs. size relationship differs between preserved and fresh material — ANCOVA (F2,38 = 59.1 p = < 0.0015) Difference most marked in alcohol-preserved samples Preserved samples ≠ fresh samples Does this hold true for HTV fauna? Fleming et al. (2011) Marine Biology

How do we resolve these issues?

The next stage….. Initial “pilot” study This study should employ : A preservation effects component Sampling of all conspicuous adult species Sampling of juvenile/larval species At least 3 of each species (6 better) and a range of sizes if possible Once collected the samples should be transferred to the surface for ID and pre-analytical processing. Environmental parameters (temp, salinity, depth, etc.)

Experimental Design • For each species: • - Identification • - Dissection for tissue sample • - Processing tissues from fresh • OR • - Preservation by freezing or EToH • - Determine if DC or LE necessary • - Stable Isotope Analysis • - Derive correction factor from results Sample Collection Pre-analytical processing decisions? Preservation Method Fresh Frozen Ethanol Species-specific pre-analytical processing Untouched Lipid-extracted De-calcified SIA Trophic Position δ15N & δ13C

Moving on……… How we proceed……… What are our questions?

Thank you for listening! Rogers et al., 2012

Nicholas E.C. Fleming, Queen’s University, Belfast