Name: basic methods for the study of reproductive ecology of fish in aquaria
Uploaded: 2017-07-20T13:00:00
Description: Watch this Scientific Journal Video about Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria at JoVE.com

We thank S. Yokoyama for assistance with collecting fish. We are also grateful to W. Kawamura for helpful advice about rearing methods. This study was supported by the Japan Society for the Promotion of Science (KAKENHI) through grants (No. 24370006 and 16K07507) awarded to T.S.

1. Collecting and Transporting Live Fish
NOTE: This protocol describes how to collect fish that possess a gas bladder, from a depth of &#8805;15 m to the surface. Rapid conveyance to the surface will induce expansion of the gas bladder by a change of pressure, which can seriously harm or kill the fish. Caution is warranted, as damage caused to the fish during this first step will negatively effect their survival and later behavior.
<ol>
	<li>Prior to diving with SCUBA, gather the following m...

<ol>
	<li>Helfman, G., Collette, B. B., Facey, D. E., Bowen, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Diversity of Fishes: Biology, Evolution, and Ecology. , (2009).</li><li>Davies, N. B., Krebs, J. R., West, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Behavioural Ecology. , (2012).</li><li>Fukuda, K., Manabe, H., Sakurai, M., Dewa, S., Shinomiya, A., Sunobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monogamous+mating+system+and+sexuality+in+the+gobiid+fish,+Trimma+marinae+(Actinopterygii:+Gobiidae).">Monogamous mating system and sexuality in the gobiid fish, Trimma marinae (Actinopterygii: Gobiidae).</a> J Ethol. 35 (1), 121-130 (2017).</li><li>Manabe, H., Matsuoka, M., Goto, K., Dewa, S., Shinomiya, A., Sakurai, M., Sunobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-directional+sex+change+in+the+gobiid+fish+Trimma+sp.:+does+size-advantage+exist?.">Bi-directional sex change in the gobiid fish Trimma sp.: does size-advantage exist?.</a> Behaviour. 145 (1), 99-113 (2008).</li><li>Sakurai, M., Nakakoji, S., Manabe, H., Dewa, S., Shinomiya, A., Sunobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-directional+sex+change+and+gonad+structure+in+the+gobiid+fish+Trimma+yanagitai.">Bi-directional sex change and gonad structure in the gobiid fish Trimma yanagitai.</a> Ichthyol Res. 56 (1), 82-86 (2009).</li><li>Sunobe, T., Nakazono, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+change+in+both+directions+by+alteration+of+social+dominance+in+Trimma+okinawae+(Pisces:+Gobiidae).">Sex change in both directions by alteration of social dominance in Trimma okinawae (Pisces: Gobiidae).</a> Ethology. 94 (4), 339-345 (1993).</li><li>Wong, M. Y., Munday, P. L., Buston, P. M., Jones, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monogamy+when+there+is+potential+for+polygyny:+tests+of+multiple+hypotheses+in+a+group-living+fish.">Monogamy when there is potential for polygyny: tests of multiple hypotheses in a group-living fish.</a> Behav Ecol. 19 (2), 353-361 (2008).</li><li>Kuwamura, T., Suzuki, S., Tanaka, N., Ouchi, E., Karino, K., Nakashima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+change+of+primary+males+in+a+diandric+labrid+Halichoeres+trimaculatus:+coexistence+of+protandry+and+protogyny+within+a+species.">Sex change of primary males in a diandric labrid Halichoeres trimaculatus: coexistence of protandry and protogyny within a species.</a> J Fish Biol. 70 (6), 1898-1906 (2007).</li><li>Winterbottom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+new+species+of+the+Trimma+tevegae+species+group+from+the+Western+Pacific+(Percomorpha:+Gobiidae).">Two new species of the Trimma tevegae species group from the Western Pacific (Percomorpha: Gobiidae).</a> Aqua J Ichthyol Aquat Biol. 10, 29-38 (2005).</li><li>Frederick, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorescent+elastomer+injection+as+a+method+for+marking+small+fish.">Evaluation of fluorescent elastomer injection as a method for marking small fish.</a> Bull Mar Sci. 61 (2), 399-408 (1997).</li><li>Olsen, E. M., V&#248;llestad, L. A. <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+visible+implant+elastomer+for+marking+age-0+brown+trout.">An evaluation of visible implant elastomer for marking age-0 brown trout.</a> N Am J Fish Manag. 21 (4), 967-970 (2001).</li><li>Leblanc, C. A., Noakes, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+implant+elastomer+(VIE)+tags+for+marking+small+rainbow+trout.">Visible implant elastomer (VIE) tags for marking small rainbow trout.</a> N Am J Fish Manag. 32 (4), 716-719 (2012).</li><li>Kinkel, M. D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+injection+into+adult+zebrafish.">Intraperitoneal injection into adult zebrafish.</a> J Vis Exp. (42), e2126 (2010).</li><li>Reavis, R. H., Grober, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrative+approach+to+sex+change:+social,+behavioural+and+neurochemical+changes+in+Lythrypnus+dalli+(Pisces).">An integrative approach to sex change: social, behavioural and neurochemical changes in Lythrypnus dalli (Pisces).</a> Acta Ethol. 2 (1), 51-60 (1999).</li><li>Milinski, M., Bakker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+sticklebacks+use+male+coloration+in+mate+choice+and+hence+avoid+parasitized+males.">Female sticklebacks use male coloration in mate choice and hence avoid parasitized males.</a> Nature. 344, 330-333 (1990).</li><li>Basolo, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+evidence+for+the+role+of+a+pre-existing+bias+in+sexual+selection.">Phylogenetic evidence for the role of a pre-existing bias in sexual selection.</a> Proc R Soc Lond B Biol Sci. 259 (1356), 307-311 (1995).</li><li>Milinski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+forging:+the+influence+of+intraspecific+competition+on+diet+selection.">Optimal forging: the influence of intraspecific competition on diet selection.</a> Behav Ecol Sociobiol. 11 (2), 109-115 (1982).</li><li>Manabe, H., Ishimura, M., Shinomiya, A., Sunobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+evidence+for+bidirectional+sex+change+in+the+polygynous+gobiid+fish+Trimma+okinawae.">Field evidence for bidirectional sex change in the polygynous gobiid fish Trimma okinawae.</a> J Fish Biol. 70 (2), 600-609 (2007).</li><li>Stammler, K. L., Corkum, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+fish+size+on+shelter+choice+and+intraspecific+interactions+by+round+gobies+Neogobius+melanostomus.">Assessment of fish size on shelter choice and intraspecific interactions by round gobies Neogobius melanostomus.</a> Environ Biol Fishes. 73 (2), 117-123 (2005).</li><li>Matsumoto, Y., Tawa, A., Takegaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+mate+choice+in+a+paternal+brooding+blenny:+the+process+and+benefits+of+mating+with+males+tending+young+eggs.">Female mate choice in a paternal brooding blenny: the process and benefits of mating with males tending young eggs.</a> Ethology. 117 (3), 227-235 (2011).</li><li>McCormick, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behaviorally+induced+maternal+stress+in+a+fish+influences+progeny+quality+by+a+hormonal+mechanism.">Behaviorally induced maternal stress in a fish influences progeny quality by a hormonal mechanism.</a> Ecology. 79 (6), 1873-1883 (1998).</li><li>Knaepkens, G., Bruyndoncx, L., Coeck, J., Eens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spawning+habitat+enhancement+in+the+European+bullhead+(Cottus+gobio),+an+endangered+freshwater+fish+in+degraded+lowland+rivers.">Spawning habitat enhancement in the European bullhead (Cottus gobio), an endangered freshwater fish in degraded lowland rivers.</a> Biodivers Conserv. 13 (13), 2443-2452 (2004).</li><li>Shiogaki, M., Dotsu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+history+of+the+gobiid+fish,+Zonogobius+boreus.">The life history of the gobiid fish, Zonogobius boreus.</a> Bulletin of the Faculty of Fisheries, Nagasaki University. 37, 1-8 (1974).</li><li>Meunier, B., Yavno, S., Ahmed, S., Corkum, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+documentation+of+spawning+and+nest+guarding+in+the+laboratory+by+the+invasive+fish,+the+round+goby+(Neogobius+melanostomus).">First documentation of spawning and nest guarding in the laboratory by the invasive fish, the round goby (Neogobius melanostomus).</a> J Great Lakes Res. 35 (4), 608-612 (2009).</li></ol>

The reproductive ecology of numerous fishes has often been revealed through experimental rearing. Especially, sex change6,8,14, mate choice15,16 and intraspecific competition7,17 have been frequent topics of detailed investigations using aquarium-kept fish. Furthermore, some results observed in aquaria have been ...

Spectacular adaptive evolution is evident in the morphology, ecology and behavior of fishes1. Especially, ecological features relating to reproduction are especially diverse, and most of these can be directly influenced by individual fitness2. To gain insight into selective pressures that have led to the evolution of unique features in different fish species, direct observation of reproductive and social behaviors using live fish is often beneficial to substantiate theoretical hypotheses.
However, continuous field observations of fish may require specialized underwater equipment and facilities that are difficult to maintain. In these cases, observations of wild-caught aquarium-reared fish can be helpful3,4,5. In addition, efficient observations of fish behaviors that are otherwise rare or difficult to observe under natural conditions can become possible by manipulating experiments in aquaria6,7,8. Rearing fish under good conditions by minimizing artificial stress and physical damage is critical for accurate ecological investigations.
The pygmy goby Trimma marinae reaches 23-25 mm total length and is distributed in the western Pacific Ocean, where it is found in quiet, sheltered bays, at depths of 9-26 m9. In this work, T. marinae is used as a model to describe a series of basic techniques for the collection of fish using the self-contained underwater breathing apparatus (SCUBA), fish transport, and eventual acclimation of the fish to aquaria for direct observation of the study species&#39; reproductive behavior and ecology.

<table border="0" cellpadding="0" cellspacing="0" width="1037">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Hand net</td>
			<td style="width:193px;">Nisso</td>
			<td style="width:108px;">AQ-17</td>
			<td style="width:544px;">Select for the target species size.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene bag</td>
			<td>San-U Fish Farm</td>
			<td>8194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rubber band</td>
			<td>ESCO Co. LTD.</td>
			<td>78-0420-64</td>
			<td>&#248; 80 mm x 6 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen cylinder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Oxgen cylinder for diving equipment suits.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elbagin</td>
			<td>Japan Pet Design Co. Ltd.</td>
			<td>75950</td>
			<td>Pafurazine F (provided from same company) is equivalent drug to Elbagin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polystyrene foam box</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette</td>
			<td>AS ONE</td>
			<td>1-8625-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rope</td>
			<td>Mizukami Kinzoku Co. LTD.</td>
			<td>95301601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Weight for diving equipment suits.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air pump</td>
			<td>KOTOBUKI</td>
			<td colspan="2">4972814 062115</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air stone</td>
			<td>KOTOBUKI</td>
			<td colspan="2">4972814 232204</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylquinoline&#160;</td>
			<td>Wako</td>
			<td>170-00376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol (99.5)</td>
			<td>Wako</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Visible implant elastomer tag kit</td>
			<td>Northwest Marine Technology</td>
			<td>N/A</td>
			<td>http://www.nmt.us/products/vie/vie.shtml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soft sponge</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVC board</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>0.3 mm thickness is easy to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>A large one, such as&#160; &#248; 160 mm and 30 mm depth, is convenient for the injection of the VIE tag.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transparent acrylic board</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UVA filtered light&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVC pipe</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>&#248; 5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterproof sheet</td>
			<td>SOMAR Corp.</td>
			<td>3EKW03</td>
			<td>The film for the plain copier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sand</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the above methods, 41, 15 and 96 individuals of T. marinae were collected in April 2014, 2015 and 2016, respectively, offshore of Amami Oshima, Kagoshima Prefecture, Japan (Table 1). In each case, 25 (61%), 14 (93%) and 91 (95%) individuals lived until depositing eggs in an aquarium. As reported in Fukuda et al.3, only one fish died before the end of the observation period in 2014, and fish spawning otherwise appeared to...

Watch this Scientific Journal Video about Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria at JoVE.com

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

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 ...

The overall goal of this procedure is to implant identifying tags in live fish without injury, to facilitate various ecological studies using wild caught fish in aquarium. This method can help answer key question in the fish behavioral ecology field;such as the reproductive and social behavior of fishes. The main advantage of this technique is that we can conduct experiment on the fish without any major negative effect on their behavior.This section shows how fish are implanted with VIE tags. If the fish cannot be held by hand because it is too small, build a surgical table to hold such fish. First cut a soft sponge into a five square centimeter section, 20 to 25 millimeters tall.Then cut a groove into the sponge that is five to 10 millimeters deep and is as wide as the fish it will hold. Next cut a piece of 0.3 millimeter thick polyvinyl chloride to precisely fit over the sponge. Then add a valley fold to the PVC, and position it into the central groove.Now transfer the assembly to a 160 millimeter dish with 30 millimeter walls, and fill the dish with water from the recovery tank until the PVC board is completely immersed. This completes the surgical table. Now proceed with tagging a fish.To begin, choose an injection site and tag color. It is safer to inject the thick muscular dorsal or caudal regions, then to inject the abdomen where internal organs may be pierced. If the study species is large enough there will be eight reasonable positions for a tag.With just one tag color and use of all eight positions, 154 unique identifying patterns can be made. For the tag color, choose the one that will best contrast the body color. Then prepare the VIE tags using the tagging kit.Mix the elastomer materials and load it into a three millimeter syringe with a 29 gauge needle. Next prepare two water tanks to use during the tag injections. One for anesthesia and one for recovery.Use an air stone to mildly circulate the water in the recovery tank. For the anesthesia tank, make a one to one mixture stock of two methylquinoline and 99.5%ethanol. Make just enough to have an 18 ppm of two methylquinoline in the tank, and mix it thoroughly into the tank water.After completing these preparations, proceed with tagging the fish. Transfer an individual fish to the anesthesia tank and cover the tank so it cannot escape. Fine tune this step according to the species.The concentration of anesthetic is an important factor. Wait until the fish does not react to water vibrations or to being touched. Do not wait for the operculum to stop moving or the fish will be at a high risk of death.When the fish is ready, remove it by hand. Use a cold water acclimated hand so the fish is not warmed too much, keeping it stronger. Lift a fish from the anesthesia tank and quickly take physical measurements.If a fish appears to recover during this time, immediately transfer it back to the anesthesia tank. If the fish is small, transfer it to the surgical table ventral side down in the PVC groove. If the fish is very small, use a binocular microscope to make the injection.If the fish happens to be large enough to hold by hand, inject it handheld. Now position the beveled side of the needle outwards, and the pointed side toward the fish. Then insert the needle subcutaneously more or less parallel to the body and very shallow, just under the skin.Ultimately the goal is to make the tag visibile, so adjust as needed. Next, inject the VIE tag while withdrawing the needle and stop the injection before the needle bevel reaches the entry point. Injecting a relatively wide area on the fish makes it easier to judge the stopping point.If the injection doesn't go well, make certain that the anesthesia is working and that the needle is quickly inserted to the appropriate depth. Once injected immediately transfer the fish to the recovery tank. If recovery takes more than two minutes, gently circulate the water by hand.After the fish recovers, return it to the rearing aquarium using a net. In the rearing aquarium, provide 10 ppm of Elbagin for three days to prevent wound infection. If the fish are housed under low visibility conditions, the VIE tags can be seen more clearly using UVA filtered light.Reproductive ecology of Trimma marinae, the Princess Pygmy Goby, was studied using VIE tags. Wild individuals collected offshore of Amami Oshima, Kagoshima Prefecture, Japan, once per year. Most survived and spawned about seven days after capture.The VIE tags were visibile and allowed identification of individual Gobys even though they are quite small. The Pygmy Goby tended to establish reproductive pairs between which most of the observed spawning occurred. Only males were observed caring for eggs.Aggressive interactions between the females suggest that the monogamous mating system may result mainly from mate guarding. When eggs were deposited on a waterproof sheet they were visible enough to be counted. After the sheet was removed, photographed and returned to its former place in the aquarium, the nesting male immediately continued paternal care.Analysis of the egg counts revealed a positive correlation between female fecundity and female body size. Whereas no difference in reproductive success was observed among differently sized males. Since developing this procedure, we have been able to perform various behavioral experiment using a lot of variety of fish more easily and accurately then we had using other fish identifying systems.

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Behavior

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.

It is unclear how top-down signals from the ventral visual stream affect movement. We developed a paradigm to test motor behavior towards a target on a 3D depth inversion illusion. Significant differences are reported in both deliberate, goal-directed movements and automatic actions under illusory and veridical viewing conditions.

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are ...

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants&#39; fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. Touch that optimally activates CT fibers is consistently rated as hedonically pleasant. Patient groups with impaired social-emotional functioning also show disordered affective touch ratings. However, relying on self-reported ratings of touch has many limitations, including recall bias and communication barriers. Here, we describe a methodological approach to study affective responses to touch via facial electromyography (EMG) that circumvents the reliance on self-report ratings. Facial EMG is an objective, quantitative, and non-invasive method to measure facial muscle activity indicative of affective responses. Responses can be assessed across healthy and patient populations without the need for verbal communication. Here, we provide two separate datasets demonstrating that CT-optimal and non-optimal touch elicit distinct facial muscle reactions. Moreover, facial EMG responses are consistent across stimulus modalities, e.g. tactile (experienced touch) and visual (observed touch). Finally, the temporal resolution of facial EMG can detect responses on timescales that supersede that of verbal reporting. Together, our data suggest that facial EMG is a suitable methodology for use in affective tactile research that can be used to supplement, or in some cases, supplant, existing measures.

We describe a protocol to assess facial muscle activity in response to experienced and observed tactile stimulation using facial electromyography.

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. ...

In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data

Subconcussive hits pose a threat to neuronal health as they have shown to induce neuronal structural damage and functional impairment without causing outward symptomology and appear to be a key contributor to an irreversible neurodegenerative disease, chronic traumatic encephalopathy (CTE). In addition, athletes can incur more than 1,000 of these hits per season. The subconcussive soccer heading model (SSHM) is a relevant, reproducible, and leading method of isolating and examining the effects of these subconcussive head impacts. By controlling variables such as ball traveling speed, the frequency of impacts, interval, ball placement to the head, as well as by measuring head impact magnitude, the SSHM provides the scientific community with a superior avenue of investigating the acute subconcussive effects on neuronal health. In this paper, we demonstrate the utility of SSHM in studying a time-course expression of neurofilament-light polypeptide (NF-L) in plasma in a repeated measures fashion. NF-L is an axonal injury marker that has previously been shown to be elevated in boxers and football players following subconcussive head trauma. Thirty-four adult aged soccer players were recruited and randomly assigned to either a soccer heading (n = 18) or kicking (n = 16) group. The heading group executed 10 headers with soccer balls projected at a velocity of 25 mph over 10 min. The kicking group followed the same protocol with 10 kicks. Plasma samples were obtained before and at 0 h, 2 h, and 24 h after heading/kicking and assessed for NF-L expressions. The heading group showed a gradual increase in plasma NF-L expression and peaked at 24 h after the heading protocol, whereas the kicking group remained consistent across the time points. These results confirmed the NF-L data from clinical field studies, encouraging the use of SSHM to validate clinical subconcussion data.

The subconcussive soccer heading model is a safe and concise methodological approach to isolate and measure the effects of subconcussive head impacts.

Subconcussive hits pose a threat to neuronal health as they have shown to induce neuronal structural damage and functional impairment without causing ...