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 materials: a hand net suitable for capturing the targeted species underwater; double polyethylene bags that are large enough for the fish; rubberbands (&#248; 80 mm x 6 mm); an oxygen cylinder for inflating the collection bags at the surface; the Elbagin, which is an antimicrobial comprises 10% sodium nifurstyrenate; a polystyrene foam box in which to place the collection bags; pipettes; and, if necessary (refer to step 1.4), a rope with a length equivalent to maximum diving depth, as well as a weight of at least 2 kg.</li>
	<li>Collect the targeted fish species using a hand net while diving; while still underwater, place the captured fish in a polyethylene bag and tie the mouth of the bag with a rubberband. If it will take &#8805;1 day to transport the fish to an aquarium, also preserve water from the natural habitat in other bags, to be used for renewing the fish-keeping water. 
	NOTE: For safety, the underwater tasks must be worked by at least two people.</li>
	<li>Surface from the collecting point with the bags of fish at a speed of no more than 1 m/min: while at a depth of 10 m or more, stop ascending for 1 - 5 min every 2 m; and from a depth of 10 m to the surface stop for 1 - 5 min every 1 m. When the captured fish are unable to maintain their buoyancy in the bag (i.e., if they float up with an expanded gas bladder while still trying to swim toward the bottom), either maintain same depth for 1 - 5 min or descend to 1 - 2 m deeper. Once the fish seem to recover their buoyancy, resume ascent to the surface. 
	NOTE: If there is a possibility that the air in SCUBA tank will run out while ascending, fasten a rope to the polyethylene bag and attach a &#8805;2 kg weight; after underwater operator has safely ascended, pull this up to the surface at the same speeds stipulated above.</li>
	<li>At beach or boat, dissolve 10 ppm Elbagin in the water of each polyethylene bag after surfacing. 
	NOTE: Although some fish may not be able maintain their buoyancy just after being brought to the surface, most can be expected to recover within one day.</li>
	<li>If the density of fish in a collecting bag is high, divide the fish among more bags to prevent from them damaging each other by rubbing against each other during transport. If the fish is an aggressive species, divide them individually between bags.</li>
	<li>Inflate the collection bag with oxygen and again seal the mouth of the bag using rubberbands. When introducing the oxygen into the bag, lower the nozzle into the bag&#39;s water to increase the dissolved oxygen content; the fully expanded bag should be 1/4 full with water.</li>
	<li>Keep the collection bags with fish in a polystyrene foam box, to maintain a stable water temperature and decrease the fish&#39;s stress under dark conditions.</li>
	<li>If it will take &#8805;1 day to transport the fish to an aquarium, once per day exchange 1/4 to 1/3 of the water in each fish-keeping bag, using water retained from the habitat to which has been added 10 ppm Elbagin, and repack each with oxygen. Each day, remove dead fish and excreta on the bottom by hand net or pipette. 
	NOTE: If airplane transportation is involved, wait at least 1 day to acclimating fish to the air-pressure on surface before transportation because two steps pressure change (from underwater to surface, and surface to upper air) in the short term may be negatively affect the survival of the fish.</li>
</ol>
2. Acclimating the Fish to an Aquarium
<ol>
	<li>Float the bag containing fish in an aquarium for 30 min to equalize the water temperature.</li>
	<li>Over a 10 min period, incrementally exchange the bag water with aquarium water, to avoid shock caused by a sudden difference in water chemistry (e.g., pH, salinity). 
	NOTE: A fish that becomes shocked by too great a difference in water chemistry may display an abnormal change in body color and/or behavior. Monitor this carefully during acclimation.</li>
	<li>Dissolve 10 ppm Elbagin in the aquarium water.</li>
	<li>Thereafter, once a day for 3 days, renew 1/3 of the aquarium water, having added 10 ppm Elbagin to the replacement water.</li>
	<li>Finally, begin to eliminate the Elbagin by exchanging half the aquarium water once a day, until the color fades.</li>
</ol>
3. Injecting a Visible Implant Elastomer (VIE) Tag to Identify Individual Fish
NOTE: In this work, individual fish are identified using VIE tags; for examples, refer to Frederick10, Olsen and V&#248;llestad11, and Leblanc and Noakes12. Also, if the study species is large enough to hold in a hand, the surgical table used in step 3.2 will not be necessary.
<ol>
	<li>Determine the injection position and tag color for each individual. The safest option is to choose an injection point in the thick muscular dorsal or caudal part of the body, and to avoid injecting it into the abdomen where internal organs may be pierced. 
	NOTE: A numbering system is described in Figure 1: if the study species is large enough for injection into 8 possible positions, this system will allow the identification of 154 individuals using a single color. Ten colors of VIE tags are available commercially. Choose the distinguishable color based on the body color of fish.</li>
	<li>For fish that are smaller than can be held in the hand, prepare a surgical table as follows (refer also to Kinkel et al.13) (Figure 2).
	<ol>
		<li>Cut out a soft sponge measuring 5 cm L x 5 cm W, and which is at least 5 - 10 mm lower than the height of a Petri dish.</li>
		<li>Cut a groove in the sponge of approximately 5 - 10 mm depth, and adjust its width to that of the approximate body width of the fish. Cut the polyvinyl chloride (PVC) board (0.3 mm thickness) to 5 cm L x 5 cm W, and bend it into a valley-fold (or M-shape).</li>
		<li>Set the grooved PVC board on top of the grooved sponge and then set the sponge into the Petri dish (&#248; 160 mm, 30 mm depth). Use water from the recovery tank to fill the Petri dish until the PVC board is adequately immersed.</li>
	</ol>
	</li>
	<li>Prepare the VIE tags according to the VIE tagging manual. Mix the elastomer materials and add the mixture to the 3-mL syringe with 29-gauge needle, as contained in the kit.</li>
	<li>Prepare two water tanks to use while performing the tag injections: one for anesthesia and one for recovery; adjust the temperature and salinity concentration of these to match the water in the rearing aquarium. Use an air pump with an air stone to mildly circulate the water in the recovery tank.</li>
	<li>Prepare an anesthesia liquid by mixing 2-methylquinoline with an equal volume of 99.5% ethanol; add this to the anesthesia tank to achieve a concentration of 18 ppm. 
	NOTE: The optimum concentration of the anesthesia liquid will depend on the species and body size of the individuals. Therefore, examine the optimum concentration for the study species in advance.</li>
	<li>Transfer individual fish to the anesthesia tank and allow them to become anesthetized: that is, wait until fish do not react to being touched or to water vibrations when the outside of the tank is tapped. As the body color changes when it is anesthetized in many fish, also monitor the body color carefully and judge whether fish is anesthetized from its changes. Because there is some possibility that fish may jump out of the anesthesia tank, keep it covered using a transparent acrylic board, at least until the fish begin to move slowly. 
	NOTE: Fine-tune the time needed to anesthetize and/or adjust the concentration of anesthesia liquid, depending on the study species and its body size. If the operculum movement is ever stopped by the anesthesia, the fish will be at a high risk of death.</li>
	<li>As fish will weaken if their body is warmed, keep fingers in the relatively cold water while anesthetizing the fish.</li>
	<li>Lift a fish from the anesthesia tank and quickly measure it or record the necessary data (total length, sex, etc.). If a fish appears to recover during measurement, immediately transfer it back to the anesthesia tank.</li>
	<li>Transfer the fish to a surgical table. Set the fish ventral side down in the PVC groove. If the body size is very small, use a binocular microscope while making the injection. If the study species is large enough to hold in a hand, inject the VIE tag while it is being held.</li>
	<li>Position the beveled side of the needle toward the outside and pointed toward the head of the fish. Insert the needle subcutaneously, more or less parallel to the body, and as near to just under the skin as possible. Adjust the insertion depth depending on the fish body size and the easiness to ultimately see the tag.</li>
	<li>Inject VIE tag while withdrawing the needle, and stop the injection before the needle bevel reaches the needle entry point (this will be easy to discern if injecting the tag in a relatively wide area).</li>
	<li>On completing the tag injection, immediately transfer the fish to the recovery tank. If recovery appears too slow, gently circulate the water by hand.</li>
	<li>After recovery, return the fish to the rearing aquarium, and continue to add 10 ppm Elbagin to the water for 3 days. 
	NOTE: Under low-visibility conditions for VIE tags, UVA filtered light will facilitate recognizing the tags.</li>
</ol>
4. Counting the Demersal Adhesive Eggs
<ol>
	<li>To create an artificial spawning nest to which the fish can deposit adhesive eggs, cut an opaque PVC pipe (&#248; 5 cm, 6 cm long) in half, perpendicular to the diameter. 
	NOTE: The above pipe size is suitable for spawning the relatively small goby T. marinae. Thus, adjust the PVC pipe size and form as is appropriate for the study species.</li>
	<li>Print a 5 mm x 5 mm grid on a waterproof sheet and cut it out to fit the inside area of the PVC pipe.</li>
	<li>Fix the waterproof sheet to the inside of the PVC pipe using a rubberband. 
	NOTE: If it is difficult for females to attach eggs on the waterproof sheet, and eggs fall from the sheet, make the sheet textured with sandpaper.</li>
	<li>Cover the aquarium bottom with a layer of sand, 1 - 2 cm thick. Insert approximately one-third of the PVC pipe obliquely into the sand, with the sheet-fixed side down.</li>
	<li>After successful spawning by the fish, remove the sheet with the egg mass from the PVC pipe and place it in a Petri dish with aquarium water. Fix a new sheet inside the PVC pipe and place this back on the bottom of the aquarium. Finally, count the eggs by photographing the egg mass.
	<ol>
		<li>Alternatively, if observations of parental egg care are to be undertaken after spawning, be careful not to bring the eggs out of the water. Rather, use a Petri dish to scoop up the eggs and sheet along with aquarium water, photograph the egg mass quickly, and then carefully reaffix the sheet with the egg mass back into the same PVC pipe before returning it all to its original position on the bottom of the aquarium. Finally, count the eggs using the picture.</li>
	</ol>
	</li>
	<li>Count the eggs using ImageJ (Figure 3). These automatic and manual cell counting methods are explained in detail in the following: Basic cell counting (The University of Chicago, Integrated Light Microscopy Core Facility, http://digital.bsd.uchicago.edu/image_j.php); Particle Analysis (http://imagej.net/Particle_Analysis); Cell Counter (https://imagej.net/Cell_Counter).
	<ol>
		<li>Otherwise, if eggs are deposited densely, estimate the number of eggs by calculating the area-density ratio: count eggs in a printed 5 x 5 mm2 grid to calculate the egg density, measure the surface area that is covered with eggs by ImageJ, and estimate the total number of eggs from above density per unit and surface area. The above surface area measurement method by ImageJ is explained in the following: Set scale&#8230; (Research Services Branch, https://imagej.nih.gov/ij/docs/menus/analyze.html); it explains how to define the spatial scale of the image; Using ImageJ to Measure Surface Area (Keene State college, Academic Technology, https://dept.keene.edu/at/2011/04/15/by-matthew-ragan/). 
		NOTE: If eggs are transparent, the black waterproof sheet is more suitable for the automatic counting method because ImageJ can recognize the outline of eggs individually.</li>
	</ol>
	</li>
</ol>

<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 authors have nothing to disclose.

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 ...

basic-methods-for-the-study-of-reproductive-ecology-of-fish-in-aquaria

Research

JoVE Journal

Behavior

11.4K Views.  Tokyo University of Marine Science and Technology. 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.

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

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 ...

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 ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...