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VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Published: August 3rd, 2016



1Department of Marine Ecology, Centre d’Estudis Avançats de Blanes (CEAB-CSIC), 2Department of Marine Biology and Oceanography, Institut de Ciències del Mar (ICM-CSIC), 3The School of Marine Science, Ruppin Academic Center

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems 1. By filtering large volumes of water 2,3, they remove and excrete particulate (plankton and detritus) and dissolved compounds 1 (and references therein) and are an important agent of benthic-pelagic coupling 4,5 and nutrient cycling 6,7. Accurately measuring the particulate and dissolved compounds removed and excreted by benthic suspension feeders (such as sponges, ascidians, polychaetes, and bivalves) is fundamental to understand their physiology, metabolism, and feeding ecology. Together with pumping rate measurements, it a....

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1. Preparatory Steps and Cleaning Procedures

  1. Cleaning solution
    1. Wear protective gear, a lab coat, and gloves at all times. Carry out these preparatory steps in a clean space free of dust and smoke.
    2. Prepare a 5-10% hydrochloric acid (HCl) solution with fresh, high quality, double distilled water.
    3. Prepare a 5% highly soluble basic mix of anionic and non-ionic surfactant solution (See Materials List) with fresh, high quality, double distilled water.
    4. .......

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Optimization of seawater collection methods

Selection of collector vials and cleaning procedure

VacuSIP-compatible collecting vessels should have a septum that allows sampling to be initiated by piercing with a syringe needle. They should withstand the elevated underwater pressure (2-3 bars at typical scuba working depths), an.......

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Preparatory steps

Collector vials for DOM and nutrient analysis

Since collector vessels may interact with dissolved micro-constituents and the sampler walls may be a substrate for bacteria growth 30-34, different vials for DOM and nutrient collection were tested. Borosilicate is not recommended for silica quantification 33,35, since glass bottles can increase the initial concentration of silica by up t.......

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We thank Manel Bolivar for his assistance in the fieldwork. We are grateful to the "Parc Natural del Montgrì, les Illes Medes i el Baix Ter" for their support to our research and sampling permissions. The underwater manipulator was designed by Ayelet Dadon-Pilosof and fabricated by Mr. Pilosof. This work was supported by the Spanish Government project CSI-Coral [grant number CGL2013-43106-R to RC and MR] and by a F.P.U fellowship from "Ministerio de Educaciòn, Cultura y Deporte (MECD)" to TM. This is a contribution from the Marine Biogeochemistry and Global Change research group funded by the Catalan Government [grant number 2014SGR1029] and ....

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Name Company Catalog Number Comments
GorillaPod, Original Joby GP000001 flexible portable tripod 
Flangeless Ferrule IDEX Health & Science  P-200X 1/16" in Blue/pk
Male Nut IDEX Health & Science  P-205X  1/16" in Green/10pk
Female to Female Luer IDEX Health & Science  P-658
Female-Male Luer IDEX Health & Science  P-655
Peek Tubing (250µm ID) IDEX Health & Science  1531 1/16" OD x 0.01in ID x 5ft lenght. Alternative ID can be used
Two component resin epoxy IVEGOR 9257 Mix well the two component resin before use
(TOC) EPA VIALS Cole -Parmer  03756-20 40 ml glass vials. Manifactured also by Thomas Scientific (ref. number 9711F09) 
HDPE VIALS Wheaton 986701 (E78620) 20 ml high-density polyethylene vials
Vacuette Z no additive Greiner bio-one 455001 pre-vacuum by the manufacturer 
Septum Sample Bottles Thomas Scientific 1755C01 250 ml glass bottles 
Septum Cap 1 Wheaton W240844SP (E7865R) 22-400 for HDPE vials 
Septum Cap 2  Wheaton W240846 (1078-5553) 24-400 for glass vials and bottles. Also manufactured by Thermo Scientific National (ref. 03-377-42)
In-line stainless steel Swinney Filter holders Pall  516-9067 13mm of diameter
PTFE Seal Washer Pall  516-8064 ring for stainless steel filter holders
TCLP Glass Filters Pall  516-9126 binder-free glass fiber filters, 13mm of diameter,  pore size 0.7µm
Polycarbonate Filter Holders  Cole -Parmer  17295 13mm of diameter
Isopore Membrane Filters Millipore GTTP01300 13mm of diameter, pore size 0.2 µm 
Contrad 2000 Solution  Decon Labs E123FH highly soluble basic mix of anionic and non-ionic surfactant solution 
Sterile Syringe Filters VWR International Eurolab S.L. 514-0061P 25mm of diameter , pore size 0.2 µm 
Fluorescein Sigma-Aldrich (old ref.28802) 46955-100G  100g 
Holdex, disposable,sterile Greiner bio-one 450263 sterile, single-use tube holder with off-center luer for Vacuette
Sterile Needles IcoGammaPlus 5160 0.7mm x 30mm
Cryovials Nalgene Nalgene V5007(Cat. No.5000-0020) 2ml 
Cryobox carton  Rubilabor M-600 145x145x55mm p/microtube 1.5 ml
Orthophosphoric Acid Sigma 79617
Paraformaldehyde Sigma P6148 500g
Glutaraldehyde Merck 8,206,031,000 25%, 1 L
Hand Vacuum Pump  Bürkle  5620-2181

  1. Gili, J. M., Coma, R. Benthic suspension feeders: their paramount role in littoral marine food webs. Trends. Ecol. Evol. 13 (8), 316-321 (1998).
  2. Reiswig, H. In situ pumping activities of tropical Demospongiae. Mar. Bio. 9, 38-50 (1971).
  3. McMurray, S., Pawlik, J., Finelli, C. Trait-mediated ecosystem impacts: how morphology and size affect pumping rates of the Caribbean giant barrel sponge. Aquat. Bio. 23 (1), 1-13 (2014).
  4. Pile, A. J., Young, C. M. The natural diet of a hexactinellid sponge: benthic-pelagic coupling in a deep-sea microbial food web. Deep-Sea Res. Pt. I. 53 (7), 1148-1156 (2006).
  5. Nielsen, T., Maar, M. Effects of a blue mussel Mytilus edulis bed on vertical distribution and composition of the pelagic food web. Mar. Ecol. Prog. Ser. 339, 185-198 (2007).
  6. De Goeij, J. M., et al. Surviving in a marine desert: the sponge loop retains resources within coral reefs. Science. 342, 108-110 (2013).
  7. Maldonado, M., Ribes, M., van Duyl, F. C. Nutrient Fluxes Through Sponges. Biology, Budgets, and Ecological Implications. Advances in Marine Biology. 62, (2012).
  8. Riisgård, H. U. On measurement of filtration rates in bivalves - the stony road to reliable data: review and interpretation. Mar. Ecol. Prog. Ser. 211, 275-291 (2001).
  9. Reiswig, H. M. Water transport, respiration and energetics of three tropical marine sponges. J. Exp. Mar. Biol. Ecol. 14, 231-249 (1974).
  10. Jiménez, E., Ribes, M. Sponges as a source of dissolved inorganic nitrogen: nitrification mediated by temperate sponges. Limnol. Oceanogr. 52 (3), 948-958 (2007).
  11. Diaz, M. C., Ward, B. Sponge-mediated nitrification in tropical benthic communities. Mar. Ecol. Prog. Ser. 156, 97-107 (1997).
  12. Ribes, M., Coma, R., Gili, J. Natural diet and grazing rate of the temperate sponge Dysidea avara (Demospongiae, Dendroceratida) throughout an annual cycle. Mar. Ecol. Prog. Ser. 176, 179-190 (1999).
  13. Jiménez, E. . Nutrient fluxes in marine sponges: methodology, geographical variability and the role of associated microorganisms. , (2011).
  14. Reiswig, H. M. Particle feeding in natural populations of three marine demosponges. Biol. Bull. 141 (3), 568-591 (1971).
  15. Reiswig, H. M. In situ pumping activities of tropical Demospongiae. Mar. Biol. 9 (1), 38-50 (1971).
  16. Yahel, G., Marie, D., Genin, A. InEx - a direct in situ method to measure filtration rates, nutrition, and metabolism of active suspension feeders. Limnol. Oceanogr-meth. 3, 46-58 (2005).
  17. Genin, A., Monismith, S. S. G., Reidenbach, M. A., Yahel, G., Koseff, J. R. Intense benthic grazing of phytoplankton in a coral reef. Limnol. Oceanogr. 54 (2), 938-951 (2009).
  18. Yahel, G., Whitney, F., Reiswig, H. M., Leys, S. P. In situ feeding and metabolism of glass sponges (Hexactinellida , Porifera) studied in a deep temperate fjord with a remotely operated submersible. Limnol. Oceanogr. 52 (1), 428-440 (2007).
  19. Wright, S. H., Stephens, G. C. Removal of amino acid during a single passage of water across the gill of marine mussels. J. Exp. Zool. 205, 337-352 (1978).
  20. Møhlenberg, F., Riisgård, H. U. Efficiency of particle retention in 13 species of suspension feeding bivalves. Ophelia. 17 (2), 239-246 (1978).
  21. Mueller, B., et al. Natural diet of coral-excavating sponges consists mainly of dissolved organic carbon (DOC). PLoS ONE. 9 (2), e90152 (2014).
  22. Gasol, J. M., Moran, X. A. G. Effects of filtration on bacterial activity and picoplankton community structure as assessed by flow cytometry. Aquat. Microb. Ecol. 16 (3), 251-264 (1999).
  23. Koroleff, F. Determination of reactive silicate. New Baltic Manual, Cooperative Research Report Series A. 29, 87-90 (1972).
  24. Murphy, J., Riley, J. P. A. Modified single solution method for the determination of phosphate in in natural waters. Anal. Chim. Acta. 27, 31-36 (1962).
  25. Shin, M. B. Colorimetric method for determination of nitrite. Ind.Eng.Chem. 13 (1), 33-35 (1941).
  26. Wood, E. D., Armstrong, F. A. J., Richards, F. A. Determination of nitrate in sea water by cadmium-copper reduction to nitrite. J. Mar. Biol. Assoc. U. K. 47 (1), 23-31 (1967).
  27. Sharp, J. H., et al. A preliminary methods comparison for measurement of dissolved organic nitrogen in seawater. Mar. Chem. 78 (4), 171-184 (2002).
  28. Sharp, J. H. Marine dissolved organic carbon: Are the older values correct. Mar. Chem. 56 (3-4), 265-277 (1997).
  29. Holmes, R. M., Aminot, A., Kerouel, R., Hooker, B. A., Peterson, B. J. A simple and precise method for measuring ammonium in marine and freshwater ecosystems. Can. J. Fish. Aquat. Sci. 56 (10), 1801-1808 (1999).
  30. Degobbis, D. On the storage of seawater samples for ammonia determination. Limnol. Oceanogr. 18 (1), 146-150 (1973).
  31. Tupas, L. M., Popp, B. N., Karl, D. M. Dissolved organic carbon in oligotrophic waters: experiments on sample preservation, storage and analysis. Mar. Chem. 45, 207-216 (1994).
  32. Yoro, S. C., Panagiotopoulos, C., Sempéré, R. Dissolved organic carbon contamination induced by filters and storage bottles. Water Res. 33 (8), 1956-1959 (1999).
  33. Zhang, J. Z., Fischer, C. J., Ortner, P. B. Laboratory glassware as a contaminant in silicate analysis of natural water samples. Water Res. 33 (12), 2879-2883 (1999).
  34. Yoshimura, T. Appropriate bottles for storing seawater samples for dissolved organic phosphorus (DOP) analysis: a step toward the development of DOP reference materials. Limnol. Oceanogr-meth. 11 (4), 239-246 (2013).
  35. Strickland, J. D. H., Parsons, T. R. . A practical handbook of seawater analysis. , (1968).
  36. Eaton, A. D., Grant, V. Freshwater sorption of ammonium by glass frits and filters: implications for analyses of brackish and freshwater. Limnol. Oceanogr. 24 (2), 397-399 (1979).
  37. Norrman, B. Filtration of water samples for DOC studies. Mar. Chem. 41 (1-3), 239-242 (1993).
  38. Carlson, C. A., Ducklow, H. W. Growth of bacterioplankton and consumption of dissolved organic carbon in the Sargasso Sea. Aquat. Microb. Ecol. 10 (1), 69-85 (1996).
  39. Grasshoff, K., Ehrhardt, M., Kremling, K. . Methods of Seawater Analysis. Second, Revised and Extended Edition. , (1999).
  40. Perea-Blázquez, A., Davy, S. K., Bell, J. J. Nutrient utilisation by shallow water temperate sponges in New Zealand. Hydrobiologia. 687 (1), 237-250 (2012).
  41. Perea-Blázquez, A., Davy, S. K., Bell, J. J. Estimates of particulate organic carbon flowing from the pelagic environment to the benthos through sponge assemblages. PLoS ONE. 7 (1), e29569 (2012).
  42. Pile, A. J., Patterson, M. R., Witman, J. D. In situ grazing on plankton <10 µm by the boreal sponge Mycale lingua. Mar. Ecol. Prog. Ser. 141, 95-102 (1996).

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