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>

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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. 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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. 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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. 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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.

<table><tbody><tr><td>10 L plastic tub</td><td>&amp;nbsp;N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>19 L pail</td><td>N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>3.00 mm scalpel</td><td>&amp;nbsp;Beaver-Vistec</td><td>377513</td><td></td></tr><tr><td>500 ml bottle</td><td>&amp;nbsp;Nalgene</td><td>&amp;nbsp;2089-0016</td><td></td></tr><tr><td>Adam scale</td><td>&amp;nbsp;Certified Material Testing Products</td><td>&amp;nbsp;BCL-LBK12A</td><td></td></tr><tr><td>Air pump</td><td>&amp;nbsp;Amazon</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Air stone</td><td>&amp;nbsp;Pentair</td><td>&amp;nbsp;ALS3</td><td></td></tr><tr><td>Dip net, 3/32 in mesh</td><td>N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Ethanol (70%)</td><td>&amp;nbsp;Sigma-Aldrich&amp;nbsp;</td><td>24102</td><td></td></tr><tr><td>Fish protector</td><td>Kordon LLC</td><td>31456</td><td></td></tr><tr><td>Foam pad</td><td>N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Gloves</td><td>Kimberly Clark&amp;nbsp;</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Mixer bar</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;F37110-1128</td><td></td></tr><tr><td>Plastic tweezer</td><td>N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Pouring dispenser gage&amp;nbsp;</td><td>Fischer Scientific</td><td>&amp;nbsp;13-683-60C</td><td></td></tr><tr><td>Scalpel handle</td><td>&amp;nbsp;Beaver-Vistec</td><td>371360</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>&amp;nbsp;S5761</td><td></td></tr><tr><td>Stirrer plate</td><td>&amp;nbsp;Corning, PC-351</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Tricaine methanesulfonate, MS-222</td><td>Western Chemical Inc.</td><td>515388</td><td>Treated fish destined for food must be held in fresh water above 10&amp;deg;C (50&amp;deg;F) for 21 days before use</td></tr><tr><td>Tubing, 6 mm</td><td>N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Developmental Biology

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Neuroscience

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Genetics

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Biochemistry

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

Behavior

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Handling and Tagging Techniques for Implanting Juvenile American Shad with a New Acoustic Microtransmitter

The use of telemetry techniques to better understand the behavior and survival of juvenile American shad (Alosa sapidissima), as they migrate through hydropower systems, has been challenging because shad are widely known to be particularly sensitive to handling. The goal of this study was to develop a tagging protocol using a new, acoustic micro transmitter that minimizes the detrimental effects of the tagging process and maximizes post-tagging survival of juvenile American shad. Limiting out-of-water handling and the use of brackish saltwater (7.5 parts per thousand) before and after tagging improved survival for shad tagged using a simple pectoral implantation method. This protocol provides a detailed, step-by-step procedure for tagging juvenile shad with acoustic transmitters. Fish tagged using this procedure and held in the laboratory for 60 days had an 81.5% survival rate, compared to 70% for their untagged counterparts. The successful tagging and handling practices developed in this study could be applied to field telemetry studies of juvenile shad and other sensitive species.

This article provides a detailed procedure for optimal handling practices and implantation of an acoustic micro transmitter into juvenile American shad. The results of our laboratory study suggest that these tagging techniques could be implemented in field studies of juvenile American shad with a high probability of survival.

The use of telemetry techniques to better understand the behavior and survival of juvenile American shad (Alosa sapidissima), as they migrate through ...

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

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

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Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Handling and Tagging Techniques for Implanting Juvenile American Shad with a New Acoustic Microtransmitter

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)