This study was funded by the U.S. Army Corps of Engineers (USACE) and the U.S. Department of Energy (DOE) Water Power Technologies Office. We appreciate the technical assistance from the following PNNL staff: Colin Brislawn, Eric Francavilla, Jill Janak, Huidong Li, and Tim Linley. We thank Kathy Kratchman from Delaware Valley Fish Company for supplying eels to PNNL and Ralph Lampman and Tyler Beals from the Yakama Nation Fisheries for supplying the lamprey. We also thank Brad Eppard, Scott Fielding, and Ricardo Walker from the USACE and Dana McCoskey and Tim Welch from the DOE. The laboratory studies were conducted at PNNL, which is operated by Battelle for the U.S. Department of Energy underContractDE-AC06-76RL01830.

PNNL is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Eels and lamprey were handled in accordance with federal guidelines for the care and use of laboratory animals, and the protocols for our study were conducted in compliance with and approved by PNNL&#39;s Institutional Animal Care and Use Committee.
1. Tag preparation (timing 22 minutes)
<ol>
	<li>Place tags in 70% ethanol solution for 20 min.</li>
	<li>Remove tags using plastic tweezers and place in small glass dish containing sterile water.</li>
	<li>Remove tags and place on a sterile pill cup.</li>
</ol>
2. Prepare anesthetic solution (timing ~ 5 minutes)
<ol>
	<li>In a small plastic cup weigh 40 g of Tricaine.</li>
	<li>Dissolve powder and tap water into a 500 mL beaker on a stirrer plate with mixer bar and then transfer to a plastic bottle to achieve an 80 g/L stock solution.</li>
	<li>Repeat the same procedure with sodium bicarbonate and add to a separate 500 mL bottle.</li>
	<li>Using a 10 L plastic tub container, fill with 5 L water (same water source as fish to be tagged).</li>
	<li>Add 6.25 mL of Tricaine anesthetic solution and 6.25 mL of sodium bicarbonate using a measuring pouring gage attached to the top of the 500 mL bottles to obtain a 100 mg/L dose to the tub.</li>
	<li>Stir to mix solution in water. 
	NOTE: Have a lid available to cover the tub to ensure lamprey do not escape while in the anesthetic solution.</li>
</ol>
3. Lamprey tagging (timing ~6 minutes)
<ol>
	<li>Place juvenile lamprey (&#62;140 mm) into anesthetic bath using a small dip net.</li>
	<li>Wait 4-5 min for lamprey to become fully demobilized (no longer swimming in solution, while maintaining a slow by steady gill movement).</li>
	<li>Use a dip net to remove lamprey, take length and weight measurements and record the unique identification code of the acoustic tag.</li>
	<li>Prepare a 1.3 cm thick closed-cell foam pad saturated with water first and then 150 &#181;L/L protective coating (see Table of Materials). The protective coating helps to counteract the disruption of mucus membranes during surgical procedure.</li>
	<li>Place the lamprey ventral side up on the prepared foam. Position a small section of tubing with regulated water supply (from an elevated water tank) to flow through the mouth region during the surgical process. This allows for respiration while fish is undergoing tag implantation.</li>
	<li>Locate the site where incision is to be made, ~20 mm posterior to the gill pores on the left or right lateral side. Use a ruler with makings or use markers placed on the foam pad for reference.</li>
	<li>Using a sterile 3.0-mm microsurgical scalpel with a 15&#176; blade (see Table of Materials) carefully make a 2.5-3 mm opening in the lateral direction ensuring that the scalpel is cutting just through the inner skin wall. See Figure 1.</li>
	<li>Place the disinfected tag anteriorly into the body cavity by hand. Apply slight pressure to the tagging site to ensure that the tag has moved into the body cavity.</li>
	<li>Place tagged lamprey into a recovery bucket containing fresh river water supplied with a fish tank oxygen pump, tubing, and air stone.</li>
	<li>Ensure that lamprey have recovered from the anesthetic and transfer to holding tank for future study.</li>
</ol>
4. Eel tagging (timing ~6 minutes)
<ol>
	<li>Replicate tag preparation and prepare anesthetic with a concentration dose of 240 mg/L for both Tricaine and sodium bicarbonate.</li>
	<li>Add 15 mL of each to the 5 L water bath.</li>
	<li>Place juvenile eels (&#62;130 mm) into anesthetic bath using a small dip net. Wait 4-5 min for eel to become fully demobilized (no longer swimming in solution while maintaining a slow but steady gill movement).</li>
	<li>Use a dip net to remove eel and place ventral side up on a closed-cell foam coated with Fish Protector.</li>
	<li>Locate the site where incision is to be made, ~25 mm to the base of the pectoral fin on the left or right lateral side</li>
	<li>Using a sterile 3.0-mm microsurgical scalpel with a 15&#176; blade carefully make a 2.5-3 mm opening in the lateral direction ensuring that the scalpel is cutting just through the inner skin wall. See Figure 2.</li>
	<li>Place the disinfected tag anteriorly into the body cavity by hand. Apply slight pressure to tagging site to ensure tag has moved into the body cavity.</li>
	<li>Place tagged eels into a recovery bucket containing fresh river water supplied with a fish tank oxygen pump, tubing, and air stone.</li>
	<li>Ensure that eel has recovered from the anesthetic and transfer to holding tank for future study.</li>
</ol>

<ol>
	<li>Mueller, R. P., 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=Retention+and+Effects+of+Miniature+Transmitters+in+Juvenile+American+Eels.">Retention and Effects of Miniature Transmitters in Juvenile American Eels.</a> Fisheries Research. 195, 52-58 (2017).</li><li>Brown, R. S., 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=An+evaluation+of+the+maximum+tag+burden+for+implantation+of+acoustic+transmitters+in+juvenile+Chinook+salmon.">An evaluation of the maximum tag burden for implantation of acoustic transmitters in juvenile Chinook salmon.</a> North American Journal of Fisheries Management. 30, 499-505 (2010).</li><li>Jepsen, N., Schreck, C., Clement, S., Thorstad, E., Spedicato, M. T., Marmulla, G., Lembo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A brief discussion of the 2% tag/bodymass rule. Aquatic telemetry: advances and applications. , 255-259 (2003).</li><li>Liedtke, T. L., 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=.">.</a> A standard operating procedure for the surgical implantation of transmitters in juvenile salmonids. , 2012-1267 (2012).</li><li>Deng, Z. D., 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=A+cabled+acoustic+telemetry+system+for+detecting+and+tracking+juvenile+salmon:+Part+2.+Three-dimensional+tracking+and+passage+outcomes.">A cabled acoustic telemetry system for detecting and tracking juvenile salmon: Part 2. Three-dimensional tracking and passage outcomes.</a> Sensors. 11 (6), 5661-5676 (2011).</li><li>Titzler, P. S., McMichael, G. A., Carter, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2010.+Autonomous+acoustic+receiver+deployment+and+mooring+techniques+for+use+in+large+rivers+and+estuaries.">2010. Autonomous acoustic receiver deployment and mooring techniques for use in large rivers and estuaries.</a> North American Journal of Fisheries Management. 30 (4), 853-859 (2010).</li><li>Renaud, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+status+of+northern+hemisphere+lampreys+(Petromyzontidae).">Conservation status of northern hemisphere lampreys (Petromyzontidae).</a> Journal of Applied Ichthyology. 13 (3), 143-148 (1997).</li><li>Dauble, D. D., Moursund, R. A., Bleich, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+behavior+of+juvenile+Pacific+lamprey,+Lampetra+tridentata.">Swimming behavior of juvenile Pacific lamprey, Lampetra tridentata.</a> Environmental Biology of Fishes. 75, 167-171 (2006).</li><li>Moursund, R. A., Dauble, D. D., Langeslay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbine+intake+diversion+screens;+investigating+effects+on+Pacific+lamprey.">Turbine intake diversion screens; investigating effects on Pacific lamprey.</a> Hydro Review. 22 (1), 40-46 (2003).</li><li>Marsh, D. M., Matthews, G. M., Achord, S., Ruehle, T. E., Sandford, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversion+of+salmonid+smolts+tagged+with+passive+integrated+transponders+from+an+untagged+population+passing+through+a+juvenile+collection+system.">Diversion of salmonid smolts tagged with passive integrated transponders from an untagged population passing through a juvenile collection system.</a> North American Journal of Fisheries Management. 19, 1142-1146 (1999).</li><li>Prentice, E. F., Flagg, T. A., McCutcheon, C. S., Parker, N. C., Giorgi, A. E., Heidinger, R. C., Jester, D. B., Prince, E. D., Winans, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+using+passive+integrated+transponder+(PIT)+tags+in+salmonids.">Feasibility of using passive integrated transponder (PIT) tags in salmonids.</a> Fish marking techniques. American Fisheries Society, Symposium . 7, 317-322 (1990).</li><li>Colotelo, A. H., 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=The+effect+of+rapid+and+sustained+decompression+on+barotrauma+in+juvenile+brook+lamprey+and+Pacific+lamprey:+Implications+for+passage+at+hydroelectric+facilities.">The effect of rapid and sustained decompression on barotrauma in juvenile brook lamprey and Pacific lamprey: Implications for passage at hydroelectric facilities.</a> Fisheries Research. 129 (130), 17-20 (2012).</li><li>Moser, M. L., Jackson, A. D., Lucas, M. C., Mueller, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+and+potential+threats+to+survival+of+migrating+lamprey+ammocoetes+and+macrophthalmia.">Behavior and potential threats to survival of migrating lamprey ammocoetes and macrophthalmia.</a> Reviews in Fish Biology and Fisheries. 25 (2), 103-116 (2015).</li><li>Dixon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology,+management,+and+protection+of+catadromous+eels.">Biology, management, and protection of catadromous eels.</a> American Fisheries. Society Symposium. 33, (2003).</li><li>ASMFC (Atlantic States Marine Fisheries Commission). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Update of the American eel stock assessment report. , 51 (2006).</li><li>DFO (Fisheries and Oceans Canada). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+potential+assessment+of+American+Eel+(Anguilla+rostrata)+in+eastern+Canada.">Recovery potential assessment of American Eel (Anguilla rostrata) in eastern Canada.</a> DFO Canadian Science Advisory Secretariat Scientific Advisory Report. , (2013).</li><li>MacGregor, R. J., 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=.">.</a> Recovery Strategy for the American Eel (Anguilla rostrata) in Ontario. , (2013).</li><li>Deng, Z., Weiland, M. A., Carlson, T. J., Eppard, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Instrumentation+of+a+Measurement+and+Calibration+System+for+an+Acoustic+Telemetry+System.">Design and Instrumentation of a Measurement and Calibration System for an Acoustic Telemetry System.</a> Sensors. 10 (4), 3090-3099 (2010).</li><li>Cook, K. V., 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=A+comparison+of+implantation+methods+for+large+PIT+tags+or+injectable+acoustic+transmitters+in+juvenile+Chinook+salmon.">A comparison of implantation methods for large PIT tags or injectable acoustic transmitters in juvenile Chinook salmon.</a> Fisheries Research. 154, 213-223 (2014).</li><li>Mesa, M. G., 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=Survival+and+Growth+of+Juvenile+Pacific+Lampreys+Tagged+with+Passive+Integrated+Transponders+(PIT)+in+Freshwater+and+Seawater.">Survival and Growth of Juvenile Pacific Lampreys Tagged with Passive Integrated Transponders (PIT) in Freshwater and Seawater.</a> Transactions of the American Fisheries Society. 141 (5), 1260-1268 (2012).</li><li>Brown, R. S., Cooke, S. J., Anderson, W. G., McKinley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+to+challenge+the+2%+rule+for+biotelemetry.">Evidence to challenge the 2% rule for biotelemetry.</a> North American Journal of Fisheries Management. 19, 867-871 (1999).</li><li>Zale, A. V., Brooke, C., Fraser, W. C. <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+surgically+implanted+transmitter+weights+on+growth+and+swimming+stamina+of+small+adult+westslope+cutthroat+trout.">Effects of surgically implanted transmitter weights on growth and swimming stamina of small adult westslope cutthroat trout.</a> Transactions of the American Fisheries Society. 134 (3), 653-660 (2005).</li><li>Geist, D. R., 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=Juvenile+Chinook+salmon+survival+when+exposed+to+simulated+dam+passage+after+being+implanted+with+a+new+microacoustic+transmitter.">Juvenile Chinook salmon survival when exposed to simulated dam passage after being implanted with a new microacoustic transmitter.</a> North American Journal of Fisheries Management. , (2018).</li><li>&#216;kland, F., Thorstad, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+on+size+and+position+of+surgically+and+gastrically+implanted+electronic+tags+in+European+silver+eel.">Recommendations on size and position of surgically and gastrically implanted electronic tags in European silver eel.</a> Animal Biotelemetry. 1, 6 (2013).</li><li>Jepsen, N., Mikkelsen, J. S., Koed, A. <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+tag+and+suture+type+on+survival+and+growth+of+brown+trout+with+surgically+implanted+telemetry+tags+in+the+wild.">Effects of tag and suture type on survival and growth of brown trout with surgically implanted telemetry tags in the wild.</a> Journal of Fish Biology. 72, 594-602 (2008).</li><li>Brown, R. S., 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=Survival+of+seaward-migrating+PIT+and+acoustic-tagged+juvenile+Chinook+salmon+in+the+Snake+and+Columbia+Rivers:+an+evaluation+of+length-specific+tagging+effects.">Survival of seaward-migrating PIT and acoustic-tagged juvenile Chinook salmon in the Snake and Columbia Rivers: an evaluation of length-specific tagging effects.</a> Animal Biotelemetry. 1, 8 (2013).</li><li>Walker, R., 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=Effects+of+a+novel+acoustic+transmitter+on+swimming+performance+and+predator+avoidance+of+juvenile+Chinook+salmon:+Determination+of+a+size+threshold.">Effects of a novel acoustic transmitter on swimming performance and predator avoidance of juvenile Chinook salmon: Determination of a size threshold.</a> Fisheries Research. 176, 48-54 (2016).</li></ol>

We have nothing to disclose.

The results of the laboratory studies demonstrate that the tagging procedure and tagging effects do not have adverse effects on fish survivability or swimming ability. Extended holding and monitoring showed that minimal tag loss occurred and was not apparent during the tag life (30 days). The tag implantation procedure was effective at placing tags in the body cavity without causing significant hemorrhaging or fungal infections at the tagging site. The duration of the entire tagging process (&#60;6 min) is beneficial in that it reduces stress associated with the fish being anesthetized. These findings characterize a new tool for juvenile lamprey and eel passage research and can be utilized in future studies. This technology will allow researchers to track lamprey and eel movements within river systems and as they approach hydroelectric dams or other structures that impede fish passage. In turn, the results can better inform management decisions at these facilities to help conserve these species throughout their juvenile life stages. The most critical steps of the tag implantation protocol include using the proper dose of anesthetic, depth and length of the incision, placement of the tags, and having the proper recovery holding tanks available. This technique has minimal limitations. Most notably, surgeon training is required to effectively make the &#60;3 mm incision and place the tags at the desired location. Additionally, the tagging process would exclude smaller sized eels and lamprey (&#60;140 mm) due to tag burden limitations. We do not recommend any modifications to the described protocols.
Alternate methods for tagging eels and lamprey include the use of PIT tags. However, PIT-tags do not actively transmit signals, therefore PIT-tagged fish can only be enumerated as they are entrained and diverted into bypass facilities, or pass close to a fixed detector10,11. Future applications of the lamprey/eel tag include the ability to tag and track populations of migrating lamprey and eels in any environment they inhabit. Additional benefits of using the lamprey/eel tag would include the ability to estimate survival, fallback rates at spillways or via turbines, travel time within reservoirs, passage delays and related behavior as fish approach dams.

Understanding fish behavior and movements near river structures, such as hydropower dams, that may impede upstream or downstream migration routes is an ongoing research need. Although numerous studies have been conducted using existing technology, there are still many research questions about juvenile eels and lamprey survival and behavior. To help address these questions, Pacific Northwest National Laboratory (PNNL) has developed a new acoustic micro transmitter specifically designed for use in juvenile eels and lampreys, called the lamprey/eel tag1. Prior to this development, existing acoustic tags were too large to be effectively implanted into the body cavities of juvenile eels and lampreys, and would result in a tag burden that exceeds accepted standards (a conservative value of tag to body weight ratio is 2% or less and other literature suggests 4-5%)2,3,4. The lamprey/eel tags emit unique coded signals at a frequency of 416.7 kHz, which are monitored via autonomous receivers (hydrophones) at fixed structures or in-river receivers5,6.
In the Columbia River Basin in the United States, awareness and concern for understanding the entire life cycle of the Pacific Lamprey (Entosphenus tridentatus) has escalated because populations have significantly declined in the past 40 years7. Construction and operation of hydroelectric facilities may negatively affect juvenile lamprey, as their declines occurred after the period of major hydroelectric development8. One potential source of mortality is dam passage when juvenile Pacific Lamprey out-migrate to the ocean9. Passive Integrated Transponders (PIT) are commonly used for migrating fish species that may pass diversion structures (e.g., juvenile bypass systems), which allows tagged fish to be enumerated10,11. However, juvenile lamprey are thought to migrate deeper in the water column than juvenile salmon and are less likely to pass through the juvenile bypass systems12. Because of the low detection probabilities and a lack of hatcheries or other concentrated sources of juvenile lamprey for PIT tagging, information on juvenile lamprey passage, survival, or behavior is very limited. Knowledge of juvenile Pacific Lamprey behavior and survival are critical for developing mitigation strategies for downstream passage, including design of bypass systems for use at hydroelectric facilities and irrigation diversion structures13.
The American eel (Anguilla rostrata) is catadromous with the adult phase migrating from freshwater toward the ocean to spawn. Their population levels have seen dramatic declines over the past several decades. Previously, they were very abundant in all major rivers flowing into the Atlantic Ocean and upstream through the St. Lawrence River to Lake Ontario; but since 1980, American eels have experienced significant declines in stock abundance ranging from 50% in Chesapeake Bay to as much as 97% in Lake Ontario14,15,16. The factors contributing to their decline include; the construction of hydroelectric dams, fragmentation and loss of habitat, and commercial harvesting17. They are currently listed as Endangered under the Ontario (Canada) Endangered Species Act. The past development of hydropower facilities along major rivers of Eastern U.S. states create obstacles that impede riverine migrations of both juveniles and adults.
The newly developed lamprey/eel acoustic tag was used in this study. The tags were manufactured at PNNL&#39;s accredited Bio-Acoustics &#38; Flow Laboratory18. The tag measures 12 mm length x 2 mm diameter, and has a weight of 0.08 g in air. Because of the tag&#39;s small overall size, it can be effectively used for implantation into anguilliform or juvenile fish using a sutureless incision, due to the small incision required (&#60;3 mm long). Additional benefits of sutureless incision include: a reduced amount of time required for surgical process, faster healing rates, and decreased possibility of infections at the implantation site19. Surgical implantation effects can vary in response to species, life stage, body cavity length, incision location, study duration, and environmental conditions20,21,22,23. In addition to size, weight is an important variable because it provides a measure of the tag burden (i.e., the weight of the tag relative to the weight of the fish). The tag burden, in association with all other aspects of the surgical process (e.g., anesthesia, handling, surgery), can have a direct effect on tag retention, survival, growth, swimming performance, or the ability of fish to avoid predation24,25,26,27.

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			<td height="20" style="height:20px;width:223px;">10 L plastic tub</td>
			<td style="width:126px;">&#160;N/A</td>
			<td style="width:166px;">&#160;N/A</td>
			<td style="width:182px;"></td>
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			<td height="20" style="height:20px;width:223px;">19 L pail</td>
			<td style="width:126px;">N/A</td>
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			<td height="20" style="height:20px;width:223px;">3.00 mm scalpel</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:223px;">500 ml bottle</td>
			<td style="width:126px;">&#160;Nalgene</td>
			<td style="width:166px;">&#160;2089-0016</td>
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			<td height="28" style="height:28px;width:223px;">Adam scale</td>
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			<td style="width:166px;">&#160;BCL-LBK12A</td>
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			<td height="20" style="height:20px;width:223px;">Air pump</td>
			<td style="width:126px;">&#160;Amazon</td>
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			<td height="20" style="height:20px;width:223px;">Dip net, 3/32 in mesh</td>
			<td style="width:126px;">N/A</td>
			<td style="width:166px;">&#160;N/A</td>
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			<td height="20" style="height:20px;width:223px;">Ethanol (70%)</td>
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			<td style="width:126px;">Kordon LLC</td>
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			<td height="20" style="height:20px;width:223px;">Foam pad</td>
			<td style="width:126px;">N/A</td>
			<td style="width:166px;">&#160;N/A</td>
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			<td height="20" style="height:20px;width:223px;">Gloves</td>
			<td style="width:126px;">Kimberly Clark&#160;</td>
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			<td height="20" style="height:20px;width:223px;">Mixer bar</td>
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			<td height="20" style="height:20px;width:223px;">Plastic tweezer</td>
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			<td style="width:166px;">&#160;N/A</td>
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			<td height="20" style="height:20px;width:223px;">Pouring dispenser gage&#160;</td>
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			<td style="width:166px;">&#160;13-683-60C</td>
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			<td style="width:166px;">&#160;S5761</td>
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			<td height="20" style="height:20px;width:223px;">Stirrer plate</td>
			<td style="width:126px;">&#160;Corning, PC-351</td>
			<td style="width:166px;">&#160;N/A</td>
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			<td height="56" style="height:56px;width:223px;">Tricaine methanesulfonate, MS-222</td>
			<td style="width:126px;">Western Chemical Inc.</td>
			<td style="width:166px;">515388</td>
			<td style="width:182px;">Treated fish destined for food must be held in fresh water above 10&#176;C (50&#176;F) for 21 days before use</td>
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			<td height="20" style="height:20px;width:223px;">Tubing, 6 mm</td>
			<td style="width:126px;">N/A</td>
			<td style="width:166px;">&#160;N/A</td>
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implantation of a new micro acoustic tag in juvenile pacific lamprey and american eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

Juvenile Lamprey Laboratory Studies
A study was conducted at PNNL in 2015 to determine the feasibility of implanting a dummy lamprey/eel tag in actively migrating juvenile Pacific Lamprey. A total of 195 Pacific Lamprey (macrophthalmia life stage) were transported from a field collection site to PNNL. Two separate tasks were conducted. The first was to determine how fish respond to tag implantation. The variables determined included tag loss, delayed mortality, and healing rates for tagged fish as compared to an untagged (control) group. For the second task swim trials were conducted with tagged and untagged fish from 10 mm size categories (120-160 mm) to determine any adverse effects associated with swimming ability. Fish weights ranged from 1.8 to 7.0 g and tag burdens ranged from 4.8 to 1.25%. An x-ray illustrating the tag location inside the body cavity is shown in Figure 31. A total of two fish dropped their tags during the first 2 days of holding and no additional dropped tags were found during the remainder of the 28-day holding period. The cumulate mortality during this period was 14.3% (7 fish) for the tagged lamprey and 9.6% (5 fish) for the control group. Individual tagged and control fish were placed in a swimming chamber and swam at a constant water velocity of 11 cm/s. The flow rate was selected based on previous laboratory testing which indicated a constant swimming motion at the lower end of the sustained swimming ability9. The mean swim time of the control group was 3.15 min (SE = 42.5 s) and the mean swim time of the implanted group was 2.3 min (SE = 30.2 s), which was not a significant difference (t = 0.958; p = 0.172). The results from the swimming performance tests showed no significant correlation between fish length and swimming duration (i.e., time to impingement) for either the tagged or control groups (Figure 4). Impingement is described as the fish no longer able to swim at the constant water velocity and rested at the downstream screen.
American Eel Laboratory Studies
To determine any possible l tag effects on swimming performance, we conducted&#160;critical swimming speed tests using Ucrit, (an index of prolonged swimming performance) for six size groups (n = 120; 113-175 mm) of untagged and tagged eels&#160;using dummy tags which measured 11.4 mm x 1.8 mm (length x diameter)1. There was no significant difference in Ucrits&#160;between tagged and untagged eels as measured in cm/s (Figure 5) or median values measured in body length/s for all tagged and untagged groups combined (Figure 6). We also conducted a prolonged holding of tagged eels (38 days) and found no mortality from the tagged groups and a low tag loss of 3.8% (1 out of 26 fish)1. Based on our laboratory results, we conclude that micro acoustic tags can be effectively implanted in juvenile American eels with no significant effects on swimming ability, long term survival and had minimal tag loss during the tag life (30 days).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59274/59274fig1.jpg" /> 
Figure 1.&#160;Photographs illustrating tagging sequence for tag insertion in juvenile Pacific Lamprey. (A) Lamprey positioned on foam pad. (B) Incision made with microsurgical scalpel. (C) Tag being implanted. (D) Lamprey post-tagging. All pictures were taken of the same lamprey (148 mm).&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59274/59274fig2.jpg" /> 
Figure 2.&#160;Photographs illustrating tagging procedure for juvenile American Eels.&#160;(A) Before incision. (B) After incision. (C) After anterior insertion of the tag. All pictures were taken of the same eel (138 mm).&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59274/59274fig3.jpg" /> 
Figure 3. X-ray images of dummy lamprey/eel tag inside the body cavity of a juvenile Pacific Lamprey.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59274/59274fig4.jpg" /> 
Figure 4. Impingement rates from swimming performance tests for control (untagged) and tagged lamprey, separated into 10 mm size categories with positive standard error bars. Sample sizes are listed in the individual bars.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59274/59274fig5.jpg" /> 
Figure 5.&#160;Box plots of critical swimming speed for size groups of eels tested in cm/s for tagged and untagged eels (10 per treatment). The lines within each box represent the median; the top and bottom lines represent the 75th and 25th percentiles, respectively; the whiskers are the top 90th and bottom 10th percentiles; and the outliers are depicted by enclosed circles.&#160;
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59274/59274fig6.jpg" /> 
Figure 6.&#160;Box plots of critical swimming speed in body length/s for tagged and untagged eels (10 per treatment). The lines within each box represent the median; the top and bottom lines represent the 75th and 25th percentiles, respectively; the whiskers are the top 90th and bottom 10th percentiles; and the outliers are depicted by enclosed circles.

Watch this Scientific Journal Video about Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel at JoVE.com

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

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10.1K Views.  Pacific Northwest National Laboratory. This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

Video: Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...