Name: rapid testing of resistance of timber to biodegradation by marine wood-boring crustaceans
Uploaded: 2022-01-29T13:00:00
Description: Watch this Scientific Journal Video about Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans at JoVE.com

Thank you to the Research Council of Norway (Oslo Regional Fund, Alcofur rffofjor 269707) and the University of Portsmouth (Faculty of Science PhD research bursary) for providing funding for the studies of Lucy Martin. Also, to Gervais S. Sawyer who provided the wood used to generate the representative results. Turpentine was provided by Prof. Philip Evans of the University of British Columbia.

1. Preparing Test Sticks
<ol>
	<li>After any treatment processes are complete, cut dry wood into test sticks to size 2 mm x 4 mm x 20 mm (Figure 1). Air dry sticks to a constant weight, under laboratory conditions. Use at least 5 replicates of each wood being tested.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62776/62776fig01.jpg" /> 
Figure 1: Tes...

<ol>
	<li>Morrell, J. J., Kutz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+wood-based+materials.">Protection of wood-based materials.</a> Handbook of environmental degradation of materials, 3rd ed. , 343-368 (2018).</li><li>Distel, D. 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US EPA - Office of prevention, pesticides and toxic substances Available from: <a target="_blank" href="https://swap.stanford.edu/20110202084/http://www.epa.gov/oppsrrd1/reregistration/REDs/cca_red.pdf">https://swap.stanford.edu/20110202084/http://www.epa.gov/oppsrrd1/reregistration/REDs/cca_red.pdf</a> (2008)</li><li>Arsenic timber treatments (CCA and arsenic trioxide) review scope document, Review series 03.1. ISSN number 1443. Australian pesticides and veterinary medicines authority Available from: <a target="_blank" href="https://apvma.gov.au/sites/default/files/publication/14296-arsenic-timber-review-scope.pdf">https://apvma.gov.au/sites/default/files/publication/14296-arsenic-timber-review-scope.pdf</a> (2003)</li><li>Commission directive 2003/2/EC of 6 January 2003 relating to restrictions on the marketing and use of arsenic (tenth adaptation to technical progress to Council Deretive 76/769/EEC). 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Wood preservatives- Determination of the protective effectiveness against marine wood borers.</a> The European Commission for Standardization (CEN). , (1992).</li><li>European Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directive+98/8/EC+concerning+the+placing+of+biocidal+products+on+the+market.">Directive 98/8/EC concerning the placing of biocidal products on the market.</a> Communication and Information Resource Centre for Administrations, Businesses and Citizens. , (2010).</li><li>Mantanis, G. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additions+to+the+taxonomy+of+the+Limnoriidae.">Additions to the taxonomy of the Limnoriidae.</a> Memoirs of the Museum of Victoria. 56 (1), 129-143 (1997).</li><li>Cookson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Australasian+species+of+Limnoriidae+(Crustacea:+Isopoda).">Australasian species of Limnoriidae (Crustacea: Isopoda).</a> Memoirs of the Museum of Victoria. 52 (2), 137 (1991).</li><li>Jones, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geographical+and+vertical+distribution+of+British+Limnoria+[Crustacea:+Isopoda].">The geographical and vertical distribution of British Limnoria [Crustacea: Isopoda].</a> Journal of the Marine Biological Association of the United Kingdom. 43 (3), 589-603 (1963).</li><li>Borges, L. M. S., Cragg, S. M., Busch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+assay+for+measuring+feeding+and+mortality+of+the+marine+wood+borer+Limnoria+under+forced+feeding+conditions:+A+basis+for+a+standard+test+method.">A laboratory assay for measuring feeding and mortality of the marine wood borer Limnoria under forced feeding conditions: A basis for a standard test method.</a> International Biodeterioration &amp; Biodegradation. 63 (3), 289-296 (2009).</li><li>BSI Standards Publication. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BS+EN+350:2016.+Durability+of+wood+and+wood-based+products+-+Testing+and+classification+of+the+durability+to+biological+agents+of+wood+and+wood-based+materials.">BS EN 350:2016. Durability of wood and wood-based products - Testing and classification of the durability to biological agents of wood and wood-based materials.</a> BSI Standards Publication. , (2016).</li><li>Menzies, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The phylogeny, systematics, distribution, and natural history of limnoria. , 196-208 (1951).</li><li>Palanti, S., Feci, E., Anichini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+four+tropical+wood+species+for+their+resistance+to+marine+borers+(Teredo+spp+and+Limnoria+spp)+in+the+Strait+of+Messina.">Comparison between four tropical wood species for their resistance to marine borers (Teredo spp and Limnoria spp) in the Strait of Messina.</a> International Biodeterioration &amp; Biodegradation. 104, 472-476 (2015).</li><li>Delgery, C. C., Cragg, S. M., Busch, S., Morgan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+epibiotic+heterotrich+ciliate+Mirofolliculina+limnoriae+and+moulting+on+the+faecal+pellet+production+by+the+wood-boring+isopods+Limnoria+tripunctata+and+Limnoria+quadripunctata.">Effects of the epibiotic heterotrich ciliate Mirofolliculina limnoriae and moulting on the faecal pellet production by the wood-boring isopods Limnoria tripunctata and Limnoria quadripunctata.</a> Journal of Experimental Marine Biology and Ecology. 334 (2), 165-173 (2006).</li><li>Morrell, J. J., Helsing, G. G., Graham, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+wood+maintenance+manual:+a+guide+for+proper+use+of+Douglas-fir+in+marine+exposures.">Marine wood maintenance manual: a guide for proper use of Douglas-fir in marine exposures.</a> Forest Research Laboratory. , (1984).</li><li>Slevin, C. R., Westin, M., Lande, S., Cragg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+and+marine+trials+of+resistance+of+furfurylated+wood+to+marine+borers.">Laboratory and marine trials of resistance of furfurylated wood to marine borers.</a> Eighth European Conference on Wood Modification. , 464-471 (2015).</li><li>Westin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+borer+resistance+of+acetylated+and+furfurylated+wood+-+results+from+up+to+16+years+of+field+exposure.">Marine borer resistance of acetylated and furfurylated wood - results from up to 16 years of field exposure.</a> International Research Group on Wood Preservation. , (2016).</li><li>Westin, M., Rapp, A., Field Nilsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+test+of+resistance+of+modified+wood+to+marine+borers.">Field test of resistance of modified wood to marine borers.</a> Wood Material Science and Engineering. 1 (1), 34-38 (2006).</li><li>Borges, L. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+wood+exposed+in+the+marine+environment:+Evaluation+of+the+hazard+posed+by+marine+wood-borers+in+fifteen+European+sites.">Biodegradation of wood exposed in the marine environment: Evaluation of the hazard posed by marine wood-borers in fifteen European sites.</a> International Biodeterioration &amp; Biodegradation. 96 (1), 97-104 (2014).</li><li>Treu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durability+and+protection+of+timber+structures+in+marine+environments+in+Europe:+An+overview.">Durability and protection of timber structures in marine environments in Europe: An overview.</a> BioResources. 14 (4), 10161-10184 (2019).</li><li>Williams, J. R., Sawyer, G. S., Cragg, S. M., Simm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+questionnaire+survey+to+establish+the+perceptions+of+UK+specifiers+concerning+the+key+material+attributes+of+timber+for+use+in+marine+and+freshwater+engineering.">A questionnaire survey to establish the perceptions of UK specifiers concerning the key material attributes of timber for use in marine and freshwater engineering.</a> Journal of the Institute of Wood Science. 17 (1), 41-50 (2005).</li><li>Purnell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carbon+footprint+of+reinforced+concrete.">The carbon footprint of reinforced concrete.</a> Advances in Cement Research. 25 (6), 362-368 (2013).</li><li>Hill, C. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+environmental+consequences+concerning+the+use+of+timber+in+the+built+environment.">The environmental consequences concerning the use of timber in the built environment.</a> Frontiers in Built Environment. 5, 129 (2019).</li><li>Mercer, T. G., Frostick, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaching+characteristics+of+CCA-treated+wood+waste:+a+UK+study.">Leaching characteristics of CCA-treated wood waste: a UK study.</a> Science of the Total Environment. 427, 165-174 (2012).</li><li>Brown, C. J., Eaton, R. A., Thorp, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chromated+copper+arsenate+(CCA)+wood+preservative+on+early+fouling+community+formation.">Effects of chromated copper arsenate (CCA) wood preservative on early fouling community formation.</a> Marine Pollution Bulletin. 42 (11), 1103-1113 (2001).</li><li>Brown, C. J., Eaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+chromated+copper+arsenate+(CCA)-treated+wood+to+non-target+marine+fouling+communities+in+Langstone+Harbour,+Portsmouth,+UK.">Toxicity of chromated copper arsenate (CCA)-treated wood to non-target marine fouling communities in Langstone Harbour, Portsmouth, UK.</a> Marine Pollution Bulletin. 42 (4), 310-318 (2001).</li><li>Brown, C. J., Albuquerque, R. M., Cragg, S. M., Eaton, R. 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Prior to selecting gribble specimens to be used in the feeding experiment, individuals should be screened to assess their suitability. There can be some variation in feeding rate between individuals due to differences in size, so only fully grown adult specimens should be selected.&#160;No significant difference between feeding rate of individuals between 1.5 mm and 3 mm length was detected by Borges et al., 200917. Female Limnoria brood their eggs, during which time have a reduced feedin...

Wood-borers can cause extensive damage to marine wooden structures, such as sea defences, piers, and aquaculture structures; the replacement or restoration of which costs billions of dollars per annum worldwide1,2,3. In order to protect these structures, timber is often treated to reduce biodegradation. However, due to the restriction of use of broad-spectrum biocides in Australia, EU, UK, and USA, in the marine environment, new modification techniques and species of wood that are naturally durable to borers are sought after4,5,6,7. Novel techniques for the preservation of wood in the marine environment require thorough testing in order to meet regulatory standards and limit environmental impacts from hazards such as leaching of any chemical preservative. For example, the European standard, EN 275, which is the current European standard from 1992, is used to evaluate wood preservation treatments against marine wood-borer damage8,9. This standard, along with other legislations against the use of biocidal compounds, such as CCA4,5,6,7 and creosote10, necessitates sustainable, non-toxic methods of wood protection and the use of naturally durable timber species to replace biocidal treatments11,12. Marine trials, such as those specified in EN 275, require long exposure periods and are thus expensive and slow to yield meaningful results. Laboratory tests, however, provide a much quicker alternative to test methods of preserving timber products against marine wood-borer attack, allowing rapid evaluation of adjustments to treatment schedules13. Results from this rapid laboratory experiment are designed to inform novel modification processes of wood and to identify timber species with natural durability to borer damage. A low feeding rate and vitality can indicate increased resistance in potential products and this information can then be fed back to industry partners to allow them to improve designs. Our method allows a nimble and rapid response, that is desirable in industry, and once promising products have been identified, results can be supplemented with those from marine trials.
Gribbles (Limnoria) are a genus of isopod crustacean in the family Limnoriidae. There are over 60 species of Limnoria worldwide13,14,15, with three common species found in the UK, Limnoria lignorum, Limnoria tripunctata and Limnoria quadripunctata16.&#160;They bore tunnels on the surface of wood that is submerged in seawater, often causing economically significant damage. Gribbles are highly abundant in coastal UK waters and are easy to maintain under laboratory conditions, making them ideal organisms for the study of wood biodegradation by marine wood-boring invertebrates. Evaluating the feeding rates and vitality of gribbles on different timber species and wood preservation methods can determine the efficacy of their resistance to biodegradation. The following protocol sets out a standard method for measuring gribble feeding rates, developed from that described by Borges and colleagues12,17, in addition to streamlining the introduction of image analysis to make the process operable in non-specialist labs. Image analysis is also used to reduce the practical limitations of manually counting large number of samples. Durability in long-term marine testing, according to the British Standard EN350-1:1994, are graded in reference to Pinus sylvestris sapwood18. In the short-term laboratory testing presented here, we use Scots pine (Pinus sylvestris L) sapwood as a control to testing heartwood of the species ekki (Lophira alata Banks ex C.F Gaertn), beech (Fagus sylvatica L), sweet chestnut (Castanea sativa Mill) and turpentine (Syncarpia glomulifera (Sm.) Nied). Average faecal pellet production and vitality among eight replicates per wood species was used as an indicator of durability. We provide illustrative data collected from a typical evaluation, using the gribble species Limnoria quadripunctata and a range of naturally durable timber species. Limnoria quadripunctata, identified by the keys provided by Menzies (1951), was selected as the optimal species for biodegradation trials due to the fact that it is the most well-studied member of the family and is well-established as a model species for use in biodegradation trials. This protocol is also applicable for testing woods of different treatments although the control used should be untreated replications of the same species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-well cell culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>150200</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable volume pipette</td>
			<td>Fisher Scientific</td>
			<td>FBE10000</td>
			<td>1-10 ml</td>
		</tr>
		<tr>
			<td>Beech</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Fagus sylvatica</td>
			<td>Taxonomic authority: L</td>
		</tr>
		<tr>
			<td>Ekki</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Lophira alata</td>
			<td>Taxonomic authority: Banks ex C. F. Gaertn.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>10098140</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>LMS LTD</td>
			<td>INC5009</td>
			<td></td>
		</tr>
		<tr>
			<td>Microporous specimen capsules</td>
			<td>Electron Microscopy Sciences</td>
			<td>70187-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Scots Pine</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Pinus sylvestris</td>
			<td>Taxonomic authority: L.</td>
		</tr>
		<tr>
			<td>Size 00000 paintbrush</td>
			<td>Hobby Craft</td>
			<td>5674331001</td>
			<td>Size 000 or 0000 also acceptable</td>
		</tr>
		<tr>
			<td>Sweet Chestnut</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Castanea sativa</td>
			<td>Taxonomic authority: Mill</td>
		</tr>
		<tr>
			<td>Turpentine</td>
			<td>P. Evans (Professor, Dept. Wood Science, University of British Columbia)</td>
			<td>Syncarpia glomulifera</td>
			<td>Taxonomic authority: (Sm.) Nied.</td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Fisher Scientific</td>
			<td>15544635</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid testing of resistance of timber to biodegradation by marine wood-boring crustaceans

Wood-boring invertebrates rapidly destroy marine timbers and wooden coastal infrastructure, causing billions of dollars of damage around the globe every year. As treatments of wood with broad spectrum biocides, such as creosote and chromated copper arsenate (CCA), are now restricted in marine use by legislation, naturally durable timber species and novel preservation methods of wood are required. These methods undergo testing in order to meet regulatory standards, such as the European standard for testing wood preservatives against marine borers, EN 275. Initial investigation of durable timbers species or wood preservative treatments can be achieved quickly and inexpensively through laboratory testing, which offers many advantages over marine field trials that are typically costly, long-term endeavours. Many species of Limnoria (gribble) are marine wood-boring crustaceans. Limnoria are ideal for use in laboratory testing of biodegradation of wood by marine wood-borers, due to the practicality of rearing them in aquaria and the ease of measuring their feeding rates on wood. Herein, we outline a standardizable laboratory test for assessing wood biodegradation using gribble.

A feeding experiment of L. quadripunctata was conducted over 20 days, using five different wood types (Scots pine (Pinus sylvestris L) sapwood, and heartwood of beech (Fagus sylvatica L), ekki (Lophira alata Banks ex C. F Gaertn), sweet chestnut (Castanea sativa Mil), and turpentine (Syncarpia glomulifera (Sm.) Neid)) (See Table of Materials), in November 2020. Eight replicate sticks were used per wood species and one specimen of Limnoria...

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Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans

This protocol presents a method for assessing the feeding rate of the wood-boring crustacean, Limnoria, by measuring faecal pellet production. This method is designed for use in non-specialist labs and has potential for incorporation into standard testing protocols, to evaluate enhanced wood durability under marine conditions.

Wood-boring invertebrates rapidly destroy marine timbers and wooden coastal infrastructure, causing billions of dollars of damage around the globe every ...

Wood is an excellent material for construction, widely used for marine structures, such as piers, jetties, wharves and coastal defenses, such as groynes. So really excellent for these structures because of the strength, the impact resistance, the ability of people to shape, repair, and modify structures, all of those things are good. However, there's a major problem that results in damage that must run into the billions of dollars per year in terms of damage to marine structures.This damage is caused either by ship worms, which tunnel through the word and create quite large tunnels and as we will see in this particular presentation, a small crustacean. So here's a tube with some of them in it just about visible, the size of a small ant, but they occur in large numbers. They erode away the surface of wood and, eventually, cause collapse.What are we going to do about that problem? Well, the traditional approach is effective. It employs broad spectrum biocides, but as their name implies, these biocides are chemicals which do not distinguish between the different sorts of organisms.So a marine structure may be emitting chemicals that are damaging to the wider environment. With these concerns in mind, legislation has shifted what is permitted for marine use in North America, in the European waters, and in Australian waters. There's a major shift which requires innovation where the methods of protecting the wood are directed specifically at the borders and are not things which are going to cause problems in the broader environment.So we are setting out in this presentation to demonstrate how we test wood protection methods against the gribble, the tiny crustacean, and what we're presenting is a rapid response test. The traditional methods of testing wood materials for use in the sea require a five-year testing period and companies need to be much more nimble than that allows them to be. So we are looking and have developed a rapid method of evaluating preservation methods.This allows us to interact with the engineers producing the new methods, allows them to modify and us to test again. So here in this presentation, we'll be looking at a rapid testing method for evaluating ways of protecting wooden structures from the wood boring crustacean, the gribble. This is a bucket laboratory assessment to test for resistance of timber to biodegradation by marine wood borers.We are using the standard method to assess the feeding rate of a wood boring crustacean, the gribble, by measuring its fecal pellet production as well as assessing its vitality and mortality. After any treatment processes are complete, cut dry wood into test sticks to size two millimeters by four millimeters by 20 millimeters. Add dry sticks to a constant weight under laboratory conditions.At least five rep occurs of each wood being tested should be used. Post wood preparation. Place sticks under mesh in a food safe plastic container inside the vacuum desiccator and replace the lid, ensuring that there is a tight seal facilitated by a coating of vacuum grease.Attach three-way valve between the tubing connecting the desiccator and pump with the third tube leading to open air. Ensure that the three-way valve is closed off to air and run the pump to achieve a vacuum of between 0.75 to minus one bar within the vacuum desiccator and hold this vacuum for 45 minutes to one hour. Submerge the open end of the third tube into a container of seawater.Switch the pump off and close the valve leading to the pump and slowly open the valve until seawater is drawn by the vacuum into the desiccator. Allow the water to flow until it fills the plastic container above the level of the mesh. Then withdraw the tube from the seawater in the container, allowing air to enter until the desiccator attends to atmospheric pressure.Keep the sticks submerged under the mesh until they sink to the bottom of the plastic container. Submerge seawater saturated test sticks in sea water contained in 50 millimeter Falcon tubes. Replace water regularly for a period of 20 days.Extract individual specimens of gribble from an infested woodblock. Use a pair of fine forceps and a thin paintbrush. Carefully peel back any wood that is covering the gribble burrow with the forceps.Once the gribble have been exposed, use a paintbrush to gently pick out individuals from underneath and deposit them in a Petri dish filled with seawater. Check the gribble under a microscope to identify the species and ensure that no damage was caused while extracting. Any females brooding eggs should be discarded as gravid females have a reduced feeding capacity.Limnoria quadripunctata can be identified under a stereo microscope by the four distinct tubercules arranged in a square pattern on the animal's pleotelson in addition to an on the fifth In well plates with wells of diameter 20 millimeters, place one test stick in five millimeters of unfiltered seawater between 32 and 35 PSU per well. Place treatments or species of woods randomly throughout the well plate. Add one Gribble per well.Twice per week, remove the test stick and each gribble, one per well from the plate and place into a freshly prepared well plate containing five millimeters of sea water per well. Use a paintbrush to gently brush off any fecal pellets from the stick before transferring and retain the fecal pallets within the original well. Plates can be kept in constant dark conditions as photo period does not have an effect on gribble feeding rate.For changing the well plate and collecting the fecal pellets, vitality of individual gribble should be assessed. If a gribble has died, this cause a vitality of one. A score of two is given to gribble that are no longer on the wood and lethargic or slowly moving.Three is given to gribble that are actively swimming or moving, but not on the wood. Gribble that are crawling on the wood's surface are given a vitality score of four. Finally, the highest score of five is given to gribble who have created burrows within the wood.Use a fine paint brush to separate any clumps so the individual pellets are visible and brush any pellets away from the very edges of the well. Take a detailed photograph under a stereo microscope at one times magnification and upload to a computer. Ensure the pellets are in focus in the background as uniform with no shadows or light reflection on the surface of the water as this can interfere with imaging.Upload a stack of images by dragging and dropping or by selecting File, Import, Image Sequence, and Browse. Do not change any parameters and then select OK.Next, use the circle tool to select the bottom section of a well containing the fecal pellets. Remove the well edges and select Edit, Clear Outside.Make the image binary by selecting Process, Make Binary. Calibrate by selecting Analyze, Set Scale, and choose the number of pixels per millimeter for your image. Count the pellets and select Analyze, Analyze Particle in the box next to the size unit squared.Then select a lower threshold that is the same as the smallest size of a pellet using the unit scale set earlier. Invert show dropdown box, select Outlines, and then tick Summarize and press OK.Convert pellet counts to pellets per day, which give an indirect measure of feeding rate. Discard data from any molting individuals on days that molting occurred.Our representative results look to the species beech, Scots pine, turpentine, Ekki, and sweet chestnut on the feeding rate and vitality of gribbles. Daily fecal pellet production was calculated and averaged among eight replicates. Counts from individuals that were molting or had previously died were not included in the averages.The two control species beech and Scots pine saw the highest fecal pellet production while the hardwood Ekkis were below it. The highest vitality of five, shown in dark blue, is seen only on beech and Scots pine wood. Mortality represented by black, a vitality of one, was highest on sweet chestnut.The majority of remaining living individuals stayed a vitality of four in sweet chestnut, Ekki, and turpentine. The benefits of laboratory trials is that we can rapidly assess really promising modified woods for their durability against marine wood boring organisms and biodegradation in the ocean. I have some woods right here that has been really heavily degraded by these wood boring organisms.If you imagine, this was quite a decent sized piece of wood and it has been really, really eaten and chewed up. We can see how damaging these organisms are. By testing woods in our laboratory, it is cheaper, quicker, and more efficient than by simply going straight to trials in the ocean and we can actually get results really, really quickly.We can start to see whether or not the organisms can destroy and eat and survive on the wood and then we can start to pick really, really promising, modified wood that can then progress towards more expensive marine trials.

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Research

JoVE Journal

Environment

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

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.

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.

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

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results. The procedure is divided into three critical steps that include survey design, data collection, and data products. The influence of scallop behavior and the survey goal of providing an independent assessment of the U.S. sea scallop resource on the survey procedure are then discussed in the context of generalizing the method. Overall, the broad applicability and flexibility of the University of Massachusetts Dartmouth School for Marine Science and Technology (SMAST) drop camera survey demonstrates the method could be generalized and applied to a variety of sessile invertebrates or habitat focused research.

Image based surveying is an increasingly practical, non-invasive method to sample the marine environment. We present the protocol of a drop camera survey that estimates the abundance and distribution of the Atlantic sea scallop (Placopecten magellanicus). We discuss how this protocol can be generalized for application to other benthic macroinvertebrates.

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...