Name: vacusip, an improved inex method for in situ measurement of particulate and dissolved compounds processed by active suspension feeders
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Description: Watch this Scientific Journal Video about VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders at JoVE.com

We thank Manel Bolivar for his assistance in the fieldwork. We are grateful to the &#34;Parc Natural del Montgr&#236;, les Illes Medes i el Baix Ter&#34; 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 &#34;Ministerio de Educaci&#242;n, Cultura y Deporte (MECD)&#34; to TM. This is a contribution from the Marine Biogeochemistry and Global Change research group funded by the Catalan Government [grant number 2014SGR1029] and ISF grant 1280/13 and BSF grant 2012089&#160;to G. Yahel.

1. Preparatory Steps and Cleaning Procedures
<ol>
	<li>Cleaning solution
	<ol>
		<li>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.</li>
		<li>Prepare a 5-10% hydrochloric acid (HCl) solution with fresh, high quality, double distilled water.</li>
		<li>Prepare a 5% highly soluble basic mix of anionic and non-ionic surfactant solution (See Materials List) with fresh, high quality, double distilled water.</li>
...

<ol>
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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...

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 also enables a quantification of the nutrient fluxes mediated by these organisms and their ecological impact on water quality as well as on ecosystem scale processes.
Choosing the appropriate method of measuring removal and production rates of particulate and dissolved compounds by&#160;suspension filter feeders is crucial for obtaining reliable data concerning their feeding activity 8. As pointed out by Riisg&#229;rd and others, inappropriate methodologies bias results, distort experimental conditions, produce incorrect estimations of ingestion and excretion of certain substances, and can lead to erroneous quantification of the nutrient fluxes processed by these organisms.
The two most frequently employed methods to measure particulate and dissolved nutrient fluxes in filter feeders involve either incubation (indirect techniques) or simultaneous collection of ambient and exhaled water (direct techniques). Incubation techniques are based on measuring the rate of change in the concentration of particulate and dissolved nutrients in the incubated water, and estimating rates of production or removal compared to adequate controls 8. However, enclosing an organism in an incubation chamber can alter its feeding and pumping behavior due to changes in the natural flow regime, due to a decline in oxygen and/or in food concentration, or due to accumulation of excretion compounds in the incubation water 7,9 (and references therein). In addition to the effects of confinement and modified water supply, a major bias of incubation techniques stems from re-filtration effects (see for example 10). Although some of these methodological problems have been overcome by using the right volume and shape of the incubation vessel 11 or with the introduction of a recirculating bell-jar system in situ12, this technique often underestimates removal and production rates. Quantifying the metabolism of dissolved compounds such as dissolved organic nitrogen (DON) and carbon (DOC) or inorganic nutrients, has proven to be especially prone to biases caused by incubation techniques 13.
In the late 60s and early 70s, Henry Reiswig 9,14,15 pioneered the application of direct techniques to quantify particle removal by giant Caribbean sponges, by separately sampling the water inhaled and exhaled by the organisms in situ. Due to the difficulty to apply Reiswig&#39;s technique on smaller suspension feeders and in more challenging underwater conditions, the bulk of research in this field was restricted to the laboratory (in vitro) employing mostly indirect incubation techniques 16. Yahel and colleagues refitted Reiswig&#39;s direct in situ technique to work in smaller-scale conditions. Their method, termed InEx 16, is based on simultaneous underwater sampling of the water inhaled (In) and exhaled (Ex) by undisturbed organisms. The different concentration of a substance (e.g., bacteria) between a pair of samples (InEx) provides a measure of the retention (or production) of that substance by the animal. The InEx technique employs open-ended tubes and relies on the excurrent jet produced by the pumping activity of the studied organism to passively replace the ambient water in the collecting tube. While Yahel and colleagues have successfully applied this technique in the study of over 15 different suspension feeders taxa (e.g., 17), the method is constrained by the high level of practice and experience required, by the minuscule size of&#160;some excurrent orifices, and by sea conditions.
To overcome these obstacles, we developed an alternative technique based on controlled suction of the sampled water through minute tubes (external diameter &#60; 1.6 mm). Our goal was to create a simple, reliable, and inexpensive device that would allow clean and controlled in situ water sampling from a very specific point, such as the excurrent orifice of benthic suspension feeders. To be effective, the method has to be non-intrusive so as not to affect the ambient flow regime or modify the behavior of the studied organisms. The device presented here is termed VacuSIP. It is a simplification of the SIP system developed by Yahel et al. (2007) 18 for ROV-based point sampling in the deep sea. The VacuSIP is considerably cheaper than the original SIP and it has been adapted for SCUBA-based work. The system was designed according to principles presented and tested by Wright and Stephens (1978) 19 and M&#248;hlenberg and Riisg&#229;rd (1978) 20 for laboratory settings.
Although the VacuSIP system was designed for in situ studies of the metabolism of benthic suspension feeders, it can also be used for laboratory studies and wherever a controlled and clean, point-source water sample is required. The system is especially useful when integration over prolonged periods (min-hours) or in situ filtrations are required. The VacuSIP has been used successfully at the Yahel lab since 2011, and has also been employed in two recent studies of nutrient fluxes mediated by Caribbean and Mediterranean sponge species 21 (Morganti et al. submitted).
The use of specific samplers, the prolonged sampling duration, and the field conditions, in which VacuSIP is applied, entail some deviations from standard oceanographic protocols for collecting, filtering, and storing samples for sensitive analytes. To reduce the risk of contamination by the VacuSIP system or the risk of modification of the sampled water by bacterial activity after collection, we tested various in situ filtration and storage procedures. Different filtering devices, collection vessels, and storing procedures were examined in order to achieve the most suitable technique for the analysis of dissolved inorganic (PO43-, NOx-, NH4+, SiO4) and organic (DOC + DON) compounds, and ultra-plankton (&#60;10 &#181;m) and particulate organic (POC + PON) sampling. To further reduce the risk of contamination, especially under field conditions, the number of handling steps was reduced to the bare minimum. The visual format in which the method is presented is oriented to facilitate reproducibility and to reduce the time required to efficiently apply the technique.
System overview 
To sample in situ&#160;pumped water from suspension feeders with exhalant orifices as small as 2 mm, the pumping activity of each specimen is first visualized by releasing filtered fluorescein dyed seawater next to the inhalant orifice(s) and observing its flow from the excurrent aperture 16 (see also Figure 2B in 18). The water inhaled and exhaled by the study specimen (incurrent and excurrent) are then simultaneously sampled with the use of a pair of minute tubes installed on custom-built manipulator or on two of the &#34;arms&#34; of an upside-down flexible portable tripod (Figure 1 and Supplementary Video 1). The water inhaled by the study organism is collected by carefully positioning the proximal end of one tube inside or near the inhalant aperture of the study organism. An identical tube is then positioned inside the excurrent orifice. This operation requires good care to avoid contact or disturbance of the animal, e.g., by sediment resuspension. To begin the sampling, a diver pierces a septum in the collecting vessel with a syringe needle attached to the distal end of each tube, allowing the external water pressure to force the sampled water into the vessel through the sampling tube. The suction is initiated by the vacuum previously created in the vials and by the pressure difference between the external water and the evacuated sample container.
To ensure a clean collection of exhaled water and to avoid accidental suction of ambient water 16, the water sampling rate needs to be kept at a significantly lower rate (&#60;10%) than the excurrent flow rate. The suction rate is controlled by the length of the tube and its internal diameter (ID). The small internal diameter also ensures a negligible dead volume (&#60; 200&#160;&#181;l per meter of tubing). Sampling over prolonged periods (minutes to hours) makes it possible to integrate the inherent patchiness of most substances of interest. To ensure that samples are adequately preserved in prolonged underwater sampling sessions as well as for transportation to the lab, an in situ filtration is recommended for sensitive analytes. The selection of sampling vessels, filtration assembly, and tubing are dictated by the study organisms and the specific research question. The protocol described below assumes that a full metabolic profile is of interest (for an overview see Figure 2). However, the modular nature of the protocol allows for easy modification to accommodate simpler or even very different sampling schemes. For a full metabolic profile, the sampling protocol should include the following steps: (1) Flow visualization; (2) Sampling ultra-plankton feeding (plankton &#60; 10 &#181;m); (3) Sampling inorganic nutrients uptake and excretion (using in-line filters); (4) Sampling dissolved organic uptake and excretion (using in-line filters); (5) Particulate feeding and excretion (using in-line filters); (6) Repeat step 2 (ultra-plankton feeding as quality check); (7) Flow visualization.
When logistically feasible, it is recommended that the metabolic profile measurements are combined with pumping rate (e.g., the dye front speed method, in 16) as well as with respiration measurements. These measurements are best taken at the beginning and end of the sampling session. For respiration measurement, underwater optodes or micro-electrodes are preferable.

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			<td height="21" style="height:21px;width:280px;">GorillaPod, Original</td>
			<td style="width:191px;">Joby</td>
			<td style="width:167px;">GP000001</td>
			<td style="width:459px;">flexible portable tripod&#160;</td>
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			<td height="21" style="height:21px;">Flangeless Ferrule</td>
			<td>IDEX Health &#38; Science&#160;</td>
			<td>P-200X</td>
			<td>1/16&#34; in Blue/pk</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Male Nut</td>
			<td>IDEX Health &#38; Science&#160;</td>
			<td>P-205X&#160;</td>
			<td>1/16&#34; in Green/10pk</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Female to Female Luer</td>
			<td>IDEX Health &#38; Science&#160;</td>
			<td>P-658</td>
			<td></td>
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			<td height="21" style="height:21px;">Female-Male Luer</td>
			<td>IDEX Health &#38; Science&#160;</td>
			<td>P-655</td>
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			<td height="21" style="height:21px;">Peek Tubing (250&#181;m ID)</td>
			<td>IDEX Health &#38; Science&#160;</td>
			<td>1531</td>
			<td>1/16&#34; OD x 0.01in ID x 5ft lenght. Alternative ID can be used</td>
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			<td height="21" style="height:21px;">Two component resin epoxy</td>
			<td>IVEGOR</td>
			<td>9257</td>
			<td>Mix well the two component resin before use</td>
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			<td>Cole -Parmer&#160;</td>
			<td>03756-20</td>
			<td>40 ml glass vials. Manifactured also by Thomas Scientific (ref. number 9711F09)&#160;</td>
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			<td height="21" style="height:21px;">HDPE VIALS</td>
			<td>Wheaton</td>
			<td>986701 (E78620)</td>
			<td>20 ml high-density polyethylene vials</td>
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			<td height="21" style="height:21px;">Vacuette Z no additive</td>
			<td>Greiner bio-one</td>
			<td>455001</td>
			<td>pre-vacuum by the manufacturer&#160;</td>
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			<td>Thomas Scientific</td>
			<td>1755C01</td>
			<td>250 ml glass bottles&#160;</td>
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			<td height="21" style="height:21px;">Septum Cap 1</td>
			<td>Wheaton</td>
			<td>W240844SP (E7865R)</td>
			<td>22-400 for HDPE vials&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Septum Cap 2&#160;</td>
			<td>Wheaton</td>
			<td>W240846 (1078-5553)</td>
			<td>24-400 for glass vials and bottles. Also manufactured by Thermo Scientific National (ref. 03-377-42)</td>
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			<td height="18" style="height:19px;">In-line stainless steel Swinney Filter holders</td>
			<td>Pall&#160;</td>
			<td>516-9067</td>
			<td>13mm of diameter</td>
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			<td height="21" style="height:21px;">PTFE Seal Washer</td>
			<td>Pall&#160;</td>
			<td>516-8064</td>
			<td>ring for stainless steel filter holders</td>
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			<td height="21" style="height:21px;">TCLP Glass Filters</td>
			<td>Pall&#160;</td>
			<td>516-9126</td>
			<td>binder-free glass fiber filters, 13mm of diameter,&#160; pore size 0.7&#181;m</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate Filter Holders&#160;</td>
			<td>Cole -Parmer&#160;</td>
			<td>17295</td>
			<td>13mm of diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopore Membrane Filters</td>
			<td>Millipore</td>
			<td>GTTP01300</td>
			<td>13mm of diameter, pore size 0.2 &#181;m&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Contrad 2000 Solution&#160;</td>
			<td>Decon Labs</td>
			<td>E123FH</td>
			<td>highly soluble basic mix of anionic and non-ionic surfactant solution&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile Syringe Filters</td>
			<td>VWR International Eurolab S.L.</td>
			<td>514-0061P</td>
			<td>25mm of diameter , pore size 0.2 &#181;m&#160;</td>
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			<td height="21" style="height:21px;">Fluorescein</td>
			<td>Sigma-Aldrich</td>
			<td>(old ref.28802) 46955-100G&#160;</td>
			<td>100g&#160;</td>
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			<td height="21" style="height:21px;">Holdex, disposable,sterile</td>
			<td>Greiner bio-one</td>
			<td>450263</td>
			<td>sterile, single-use tube holder with off-center luer for Vacuette</td>
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			<td height="21" style="height:21px;">Sterile Needles</td>
			<td>IcoGammaPlus</td>
			<td>5160</td>
			<td>0.7mm x 30mm</td>
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			<td height="21" style="height:21px;">Cryovials Nalgene</td>
			<td>Nalgene</td>
			<td>V5007(Cat. No.5000-0020)</td>
			<td>2ml&#160;</td>
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			<td height="21" style="height:21px;">Cryobox carton&#160;</td>
			<td>Rubilabor</td>
			<td>M-600</td>
			<td>145x145x55mm p/microtube 1.5 ml</td>
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			<td height="21" style="height:21px;">Orthophosphoric Acid</td>
			<td>Sigma</td>
			<td>79617</td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>500g</td>
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			<td height="21" style="height:21px;">Glutaraldehyde</td>
			<td>Merck</td>
			<td>8,206,031,000</td>
			<td>25%, 1 L</td>
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			<td height="21" style="height:21px;">Hand Vacuum Pump&#160;</td>
			<td>B&#252;rkle&#160;</td>
			<td>5620-2181</td>
			<td></td>
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vacusip, an improved inex method for in situ measurement of particulate and dissolved compounds processed by active suspension feeders

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.

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...

Watch this Scientific Journal Video about VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders at JoVE.com

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

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.

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

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

The overall goal of this method is to clean the collector of water inhaled and exhaled by Benthic suspension feeders under natural conditions so that the particulate and dissolved compounds they removed and excreted can be quantified. When preparing collection vessels for dissolved organics and ammonia analysis, use new pre-cleaned EPA vials. For sampling other nutrients, use EPA glass vials.Use HDPE vials for silicate. Rinse the vials and caps with high quality double distilled water. Then replace punctured silicon septum with new ones and soak the vials and caps in 10%hydrochloric acid for at least three days followed by three rinses with high quality double distilled water.To further prepare the glass vials for nutrients, combust them at 450 degrees Celsius for four hours and allow them to cool in the furnace. Then attach the caps and wrap the vials in aluminum foil. Next mix DDW with 0.2 micron filtered surfactant to prepare the five percent solution.Now place a combusted binder free glass fiber filter inside the cleaned inline stainless steel swinnex filter holder. Then use an acid-cleaned syringe to run 100 milliliters of the filtered surfactant solution through the entire assembly. Rinse the assembly clean using 100 milliliters of high quality double distilled water.If the filter assembly is for sampling silica, then prepare it with a polycarbonate filter and run 30 milliliters of five percent hydrochloric acid through it followed by 30 milliliters of high quality double distilled water. Assemble the system for underwater work using PEEK tubing. Use a sharp knife or PEEK cutter to prepare the required lengths.Fit each tube with a male luer connector attached to a syringe needle. Make sure that the flat side of the blue flangeless ferrule is aligned with the end of the tube, then tighten the green nut by hand. Then, using insulating tape, secure the PEEK tubing to the tripod arms for a custom built manipulator.Next, attach capped disposable syringe needles to the male luer connectors. Clearly label the sampling gear and color code the inhale and exhale sampling components such as with blue tape for in and yellow for out. Similarly, color code the sampling vessels to better identify pairs of vessels that are sequentially numbered.Now evacuate the sampling vessels before the dive, ideally with a good vacuum pump. A standard Lyophilizer provides a high vacuum and works as well. Finally line up all the sampling vessels as needed for the dive.Plan all sampling steps out in advance and pack accordingly. First confirm whether the specimen is pumping by releasing filtered fluorescing dye next to the inhalant orifice and confirm that the dye is emerging through the exhale end orifice. Next install the VacuSIP device and make sure it is stable.Then place the inhalant sampling tube within the inhalant orifice or very close to it, making certain that the inhalant tube is not in the proximity of any other exhalant orifices. Next carefully direct the exhalant sampling tube towards the exhalant syphon and very gently insert it one to five millimeters into the syphon. Take great care not to make contact with or otherwise disturb the sampled organism.Then secure the sampling assembly in close working proximity. If you use the customized manipulator, take the same care in positioning the inhalant and exhalant tube. Ultra-plankton sampling requires no filter.To sample ultra-plankton use the needle to pierce the inhaled and exhaled evacuated tube's septum. Check that the water is dripping and collect two to six milliliters of water from each tube. For silicates, install the inline stainless polycarbonate filter holders with the polycarbonate membranes between the needle and the luer male connector at the distill end of the tube.Then pierce the septum cap of the prepared HDPE vials to start sampling. To sample dissolved organics and inorganics, have three pairs of vials ready. One for nitrates and phosphates, one for ammonia, and a third pair for dissolved organics.To sample dissolved organics or nutrients, replace the polycarbonate filter assembly with a pre-cleaned inline stainless steel filter holder that contains a pre-combusted glass filter. Then pierce the septum cap of an EPA glass vial to start sampling. Dissolved inorganics were collected in new EPA glass vials cleaned by the manufacturer or re-used EPA vials cleaned according to the described protocol.The recycled vials reported higher ammonia concentrations but other values were similar. Stainless steel filter holders and polycarbonate inline Swinnex filter holders were compared by filtering pure water and testing for dissolved organic and inorganic nutrients. Polycarbonate membrane and pre-combusted glass fiber filter were tested with each holder.The combination of stainless steel filter holder and combusted glass fiber filter worked best. Cell counts were made with the flow cytometer to test the sample quality. When in-ex sample pairs are properly collected, most cells were removed from the exhaled water.In contrast, improper collection did not garner the same result. When the VacuSIP is properly employed, patterns of removal or excretion were evident for most analysed compounds. The measurements seen here were taken from a mediterranean sponge.

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