Name: collecting marine gnathiid isopod fish parasites with light traps
Uploaded: 2023-09-25T14:35:00
Description: Watch this Scientific Journal Video about Collecting Marine Gnathiid Isopod Fish Parasites with Light Traps at JoVE.com

Funding was provided by the US National Science Foundation (NSF OCE&#160;2023420 and DEB 2231250, P. Sikkel PI). We thank the municipality of Dumaguete City, Negros Oriental, Philippines, for permission to conduct this study. We also thank the many volunteers for their field assistance and the staff and our colleagues at the Silliman University Institute for Environmental and Marine Sciences for their support.

Collection of samples was permitted by the Department of Agriculture-Bureau of Fisheries and Aquatic Resources (0154-18 DA-BFAR) in accordance with Philippine laws and regulations (RA 9147; FAO 233) and approved by the Silliman University (SU) animal ethics committee.
1. Light Traps
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	<li>Construction
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		<li>Construct light traps from commercial polyvinyl chloride (PVC) tubes originally designed for plumbing. Use 10-15&#160;cm diameter PVC cut to 30-40 cm length (<s...

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	<li>Sikkel, P. C., Welicky, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecological+significance+of+parasitic+crustaceans.">The ecological significance of parasitic crustaceans.</a> Parasitic Crustacea. 17 (17), 421-477 (2019).</li><li>Svavarsson, J., Bruce, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+gnathiid+isopod+crustaceans+(Cymothoida)+from+Heron+Island+and+Wistari+Reef,+southern+Great+Barrier+Reef.">New gnathiid isopod crustaceans (Cymothoida) from Heron Island and Wistari Reef, southern Great Barrier Reef.</a> Zootaxa. 4609 (1), 4609 (2019).</li><li>Shodipo, M. O., Sikkel, P. C., Smit, N. J., Hadfield, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+record+and+molecular+characterisation+of+two+Gnathia+species+(Crustacea,+Isopoda,+Gnathiidae)+from+Philippine+coral+reefs,+including+a+summary+of+all+Central-Indo+Pacific+Gnathia+species.">First record and molecular characterisation of two Gnathia species (Crustacea, Isopoda, Gnathiidae) from Philippine coral reefs, including a summary of all Central-Indo Pacific Gnathia species.</a> International Journal for Parasitology: Parasites and Wildlife. 14, 355-367 (2021).</li><li>Losey, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+symbiosis+in+Puerto+Rico+with+comparison+to+the+tropical+Pacific.">Cleaning symbiosis in Puerto Rico with comparison to the tropical Pacific.</a> 4 (4), 960-970 (1974).</li><li>Grutter, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+infestation+increases+on+coral+reefs+without+cleaner+fish.">Parasite infestation increases on coral reefs without cleaner fish.</a> Coral Reefs. 37, 15-24 (2018).</li><li>Smit, N. J., Davies, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+curious+life-style+of+the+parasitic+stages+of+gnathiid+isopods.">The curious life-style of the parasitic stages of gnathiid isopods.</a> Advances in Parasitology. 58. , 289-391 (2004).</li><li>Tanaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+history+of+gnathiid+isopods-current+knowledge+and+future+directions.">Life history of gnathiid isopods-current knowledge and future directions.</a> Plankton and Benthos Research. 2 (1), 1-11 (2007).</li><li>Sikkel, P. C., Schaumburg, C. S., Mathenia, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diel+infestation+dynamics+of+gnathiid+isopod+larvae+parasitic+on+Caribbean+reef+fish.">Diel infestation dynamics of gnathiid isopod larvae parasitic on Caribbean reef fish.</a> Coral Reefs. 25, 683-689 (2006).</li><li>Santos, T. R. N., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habitat+associations+of+fish-parasitic+gnathiid+isopods+in+a+shallow+reef+system+in+the+central+Philippines.">Habitat associations of fish-parasitic gnathiid isopods in a shallow reef system in the central Philippines.</a> Marine Biodiversity. 4, 83-96 (2019).</li><li>Nagel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vision+in+host-finding+behaviour+of+the+ectoparasite+Gnathia+falcipenis+(Crustacea).">The role of vision in host-finding behaviour of the ectoparasite Gnathia falcipenis (Crustacea).</a> Isopoda). Marine and Freshwater Behaviour and Physiology. 42 (1), 31-42 (2009).</li><li>Sikkel, P. C., Sears, W. T., Weldon, B., Tuttle, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+field+test+of+host-finding+mechanisms+in+a+Caribbean+gnathiid+isopod.">An experimental field test of host-finding mechanisms in a Caribbean gnathiid isopod.</a> Marine Biology. 158, 1075-1083 (2011).</li><li>Vondriska, C., Dixson, D. L., Packard, A. J., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+susceptible+host+fishes+exhibit+similar+chemo-attractiveness+to+a+common+coral+reef+ectoparasite.">Differentially susceptible host fishes exhibit similar chemo-attractiveness to a common coral reef ectoparasite.</a> Symbiosis. 81 (3), 247-253 (2020).</li><li>Grutter, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+infection+rather+than+tactile+stimulation+is+the+proximate+cause+of+cleaning+behaviour+in+reef+fish.">Parasite infection rather than tactile stimulation is the proximate cause of cleaning behaviour in reef fish.</a> Proceedings of the Royal Society of London. Series B: Biological Sciences. 268 (1474), 1361-1365 (2001).</li><li>Sikkel, P. C., Cheney, K. L., C&#244;t&#233;, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+evidence+for+ectoparasites+as+a+proximate+cause+of+cleaning+interactions+in+reef+fish.">In situ evidence for ectoparasites as a proximate cause of cleaning interactions in reef fish.</a> Animal Behaviour. 68 (2), 241-247 (2004).</li><li>Sikkel, P. C., Herzlieb, S. E., Kramer, 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=Compensatory+cleaner-seeking+behavior+following+spawning+in+female+yellowtail+damselfish.">Compensatory cleaner-seeking behavior following spawning in female yellowtail damselfish.</a> Marine Ecology Progress Series. , 1-11 (2005).</li><li>Triki, Z., Grutter, A. S., Bshary, R., Ros, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+short-term+exposure+to+ectoparasites+on+fish+cortisol+and+hematocrit+levels.">Effects of short-term exposure to ectoparasites on fish cortisol and hematocrit levels.</a> Marine Biology. 163, 1-6 (2016).</li><li>Hayes, P. M., Smit, N. J., Grutter, A. S., Davies, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+response+of+a+captive+blackeye+thicklip,+Hemigymnus+melapterus+(Bloch),+from+Lizard+Island,+Australia,+exposed+to+juvenile+isopods+Gnathia+aureamaculosa+Ferreira+&amp;+Smit.">Unexpected response of a captive blackeye thicklip, Hemigymnus melapterus (Bloch), from Lizard Island, Australia, exposed to juvenile isopods Gnathia aureamaculosa Ferreira &amp; Smit.</a> Journal of Fish Diseases. 34 (7), 563-566 (2011).</li><li>Grutter, A. S., Pickering, J. L., McCallum, H., 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=Impact+of+micropredatory+gnathiid+isopods+on+young+coral+reef+fishes.">Impact of micropredatory gnathiid isopods on young coral reef fishes.</a> Coral Reefs. 27 (3), 655-661 (2008).</li><li>Artim, J. M., Sellers, J. C., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropredation+by+gnathiid+isopods+on+settlement-stage+reef+fish+in+the+eastern+Caribbean+Sea.">Micropredation by gnathiid isopods on settlement-stage reef fish in the eastern Caribbean Sea.</a> Bulletin of Marine Science. 91 (4), 479-487 (2015).</li><li>Sellers, J. C., Holstein, D. M., Botha, T. L., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+and+sublethal+impacts+of+a+micropredator+on+post-settlement+Caribbean+reef+fishes.">Lethal and sublethal impacts of a micropredator on post-settlement Caribbean reef fishes.</a> Oecologia. 189, 293-305 (2019).</li><li>Allan, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+infection+directly+impacts+escape+response+and+stress+levels+in+fish.">Parasite infection directly impacts escape response and stress levels in fish.</a> Journal of Experimental Biology. 223 (16), (2020).</li><li>Spitzer, C. A., Anderson, T. W., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habitat+associations+and+impacts+on+a+juvenile+fish+host+by+a+temperate+gnathiid+isopod.">Habitat associations and impacts on a juvenile fish host by a temperate gnathiid isopod.</a> International Journal for Parasitology: Parasites and Wildlife. 17, 65-73 (2022).</li><li>Sikkel, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocturnal+migration+reduces+exposure+to+micropredation+in+a+coral+reef+fish.">Nocturnal migration reduces exposure to micropredation in a coral reef fish.</a> Bulletin of Marine Science. 93 (2), 475-489 (2017).</li><li>Artim, J. M., Hook, A., Grippo, R. S., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predation+on+parasitic+gnathiid+isopods+on+coral+reefs:+a+comparison+of+Caribbean+cleaning+gobies+with+non-cleaning+microcarnivores.">Predation on parasitic gnathiid isopods on coral reefs: a comparison of Caribbean cleaning gobies with non-cleaning microcarnivores.</a> Coral Reefs. 36, 1213-1223 (2017).</li><li>Artim, J. M., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+coral+repels+a+common+reef+fish+ectoparasite.">Live coral repels a common reef fish ectoparasite.</a> Coral Reefs. 32, 487-494 (2013).</li><li>Paula, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+corals+on+the+abundance+of+a+fish+ectoparasite+in+the+Great+Barrier+Reef.">The role of corals on the abundance of a fish ectoparasite in the Great Barrier Reef.</a> Coral Reefs. 40, 535-542 (2021).</li><li>Sikkel, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+abundance+of+fish-parasitic+gnathiid+isopods+associated+with+warm-water+bleaching+events+on+the+northern+Great+Barrier+Reef.">Changes in abundance of fish-parasitic gnathiid isopods associated with warm-water bleaching events on the northern Great Barrier Reef.</a> Coral Reefs. 38 (4), 721-730 (2019).</li><li>Shodipo, M. O., Duong, B., Graba-Landry, A., Grutter, A. S., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+acute+seawater+temperature+increase+on+the+survival+of+a+fish+ectoparasite.">Effect of acute seawater temperature increase on the survival of a fish ectoparasite.</a> In Oceans. 1 (4), (2020).</li><li>Artim, J. M., Nicholson, M. D., Hendrick, G. C., Brandt, M., Smith, T. B., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+a+cryptic+generalist+parasite+reflects+degradation+of+an+ecosystem.">Abundance of a cryptic generalist parasite reflects degradation of an ecosystem.</a> Ecosphere. 11 (10), (2020).</li><li>Richardson, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+continuous+plankton+recorder+data.">Using continuous plankton recorder data.</a> Progress in Oceanography. 68 (1), 27-74 (2006).</li><li>McLeod, L. E., Costello, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+traps+for+sampling+marine+biodiversity.">Light traps for sampling marine biodiversity.</a> Helgoland Marine Research. 71 (1), 1-8 (2017).</li><li>Artim, J. M., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+sampling+methodologies+and+estimation+of+population+parameters+for+a+temporary+fish+ectoparasite.">Comparison of sampling methodologies and estimation of population parameters for a temporary fish ectoparasite.</a> International Journal for Parasitology: Parasites and Wildlife. 5 (2), 145-157 (2016).</li><li>Pag&#225;n, J. A., Ver&#237;ssimo, A., Sikkel, P. C., Xavier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hurricane-induced+disturbance+increases+genetic+diversity+and+population+admixture+of+the+direct-brooding+isopod,+Gnathia+marleyi.">Hurricane-induced disturbance increases genetic diversity and population admixture of the direct-brooding isopod, Gnathia marleyi.</a> Scientific reports. 10 (1), (2020).</li><li>Doherty, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-traps:+selective+but+useful+devices+for+quantifying+the+distributions+and+abundances+of+larval+fishes.">Light-traps: selective but useful devices for quantifying the distributions and abundances of larval fishes.</a> Bulletin of Marine Science. 41, 423-431 (1987).</li><li>Jones, C. M., Nagel, L., Hughes, G. L., Cribb, T. H., Grutter, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+specificity+of+two+species+of+Gnathia+(Isopoda)+determined+by+DNA+sequencing+blood+meals.">Host specificity of two species of Gnathia (Isopoda) determined by DNA sequencing blood meals.</a> International Journal for Parasitology. 37 (8-9), 927-935 (2007).</li><li>Hendrick, G. C., Dolan, M. C., McKay, T., Sikkel, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+DNA+integrity+within+blood+meals+of+hematophagous+larval+gnathiid+isopods+(Crustacea).">Host DNA integrity within blood meals of hematophagous larval gnathiid isopods (Crustacea).</a> Isopoda, Gnathiidae). Parasites &amp; Vectors. 12 (1), 1-9 (2019).</li></ol>

Traditional light traps, such as those used for collecting larval fishes, are large and are suspended in water column34. In contrast, the light traps described here are small and deployed on the sea bottom. These traps can be easily transported and rapidly deployed. They can be placed by breath-hold (free)-diving in shallow sites (as in this study) or on scuba in deeper sites, and attract both fed and unfed 
juvenile stages.
Variations of the benthic light traps de...

Parasitic crustaceans are important in the ecology and life histories of reef fishes. The biomass and energy they remove from their hosts are considerable and influence behavior, physiology, and survivorship1. Gnathiid isopod crustaceans represent the most prominent group of fish parasites in tropical and subtropical reef systems, where they are both abundant and diverse2,3 and are the primary food item of cleaner fishes4,5. Gnathiids are generally 1-3 mm in size. They have unusual life histories in which only the three juvenile stages feed on the blood and body fluids of fishes6,7. They are most active at night8,9, and while vision appears to play some role, host-finding10 relies heavily on olfactory cues to find hosts11,12. Each of the three juvenile feeding stages feeds on a single host fish, with each feed separated by a molting phase. After the final feed, third-stage larvae metamorphose into non-feeding adults, which reproduce and then die. Given that feeding requires only brief association with the host, while each inter-feeding interval lasts days, gnathiids spend most of their life free-living in the benthos.
Gnathiids impact hosts in multiple ways1. Aside from their role as drivers of interactions between cleaner fish and clients13,14,15, gnathiids can increase cortisol levels and decrease hematocrit in adult fish hosts16 and in high numbers, can even cause death17. For juvenile fish, even a single gnathiid can be fatal18,19,20, and even if the fish survives, its ability to compete for space and escape predators is compromised20,21,22. Avoiding gnathiids may even constitute one of the benefits of nocturnal migration in some reef fishes23.
In addition to cleaner fishes, gnathiid populations can be impacted by other micro carnivorous fishes24, as well as corals25,26. Ocean warming and the associated loss of live corals appear to have opposing impacts on gnathiids27,28,29.
Given their clear ecological importance and the likely influence of anthropogenic environmental change on their populations, there are compelling reasons to include them in ecological studies of coral reefs. However, their unique life history and the small number of researchers who study them create a barrier to the development, implementation, and dissemination of reliable, reproducible sampling methods to collect them for research.
Light traps have long been used to collect small marine organisms at night30,31. They take advantage of and are based on the fact that many nocturnally active organisms, including arthropods, are attracted to light. Traditionally they have been used to collect planktonic organisms in the water column30. However, the basic principles can be applied to collecting free-swimming organisms that are active near the benthos. Here we present a light trapping method adapted for collecting free-living stages of gnathiid isopods near the ocean bottom in remote coral reef environments such as the Philippines. For collecting in remote areas, these light traps (Figure 1) offer some advantages over other methods developed for collecting these organisms32. They are highly portable and durable, requiring only three parts, which are easily obtainable and inexpensive. They are also negatively buoyant, as when deployed, they are completely filled with seawater. Because they depend on light for attraction, they are only effective at night for collecting nocturnally active species. They also attract more than the target species, requiring sorting of the samples under a dissecting scope to obtain the target organisms. Three methods have thus far been used by our team and collaborators to collect gnathiids in coral reef systems throughout the world32. These include emergence traps, live fish-baited traps, and light traps, each with advantages and limitations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buckets, small sample containers</td>
			<td></td>
			<td>hardware store</td>
			<td></td>
		</tr>
		<tr>
			<td>Funnels</td>
			<td>Supplier No. 2209-03</td>
			<td>Funnels: AMERICAN SCIENTIFIC LLC SE&#160;-&#160;75 mm (3&#8221;)&#160;</td>
			<td>https://us.vwr.com/store/product/8884369/plastic-funnels</td>
		</tr>
		<tr>
			<td>Main body of light traps (made from commercially available PVC sanitarty pipes)</td>
			<td>(SKU 145640)&#160;</td>
			<td>Alasco Sanitary uPVC Pipes Series 1000 107mm/4&#39;&#160;</td>
			<td>https://alascopvcpipes.com/product/alasco-standard-sanitary-upvc-pipe-series-1000/.&#160; This brand can be found in the Philippines. Other simular brands can also be used</td>
		</tr>
		<tr>
			<td>Modeling clay&#160;</td>
			<td></td>
			<td>Can be found in art suppliy and childreans toy stores</td>
			<td>To seal the funnel after retreival</td>
		</tr>
		<tr>
			<td>Plankton mesh (50-100 &#181;m)</td>
			<td></td>
			<td>any reputable brand and source</td>
			<td>https://www.adkinstruments.in/products/plankton-nets-in-various-mesh-size-1633936883</td>
		</tr>
		<tr>
			<td>Screw on lids for the light trap</td>
			<td></td>
			<td>Alasco&#160; Sanitary &#160;Clean-Out&#160; 4&#34;</td>
			<td>https://alascopvcpipes.com/product/alasco-standard-sanitary-upvc-clean-out/. This brand can be found in the Philippines. Other simular brands can also be used</td>
		</tr>
		<tr>
			<td>Scuba/snorkel equipment</td>
			<td></td>
			<td>any reputable brand and source</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo-microscopes</td>
			<td></td>
			<td>Scientific suppliers</td>
			<td></td>
		</tr>
		<tr>
			<td>Underwater touches</td>
			<td></td>
			<td>Princeton Tec Ecoflare or&#160;Fantasea Nanospotter 6023</td>
			<td></td>
		</tr>
	</tbody>
</table>

collecting marine gnathiid isopod fish parasites with light traps

A method to collect marine gnathiid isopod fish parasites with the use of light traps is presented. Gnathiid isopods are a major group of marine fish parasites that feed on blood and fluid from host fishes, mostly at night. Like ticks and mosquitos on land, they associate only temporarily with their host and spend most of their life free-living in the benthos. Given their high mobility and transient and predominantly nocturnal association with hosts, they cannot easily be collected by capturing free-living hosts. However, they are readily attracted to underwater light sources, creating the opportunity to collect them in light traps. Here the design and individual steps involved in the deployment and processing of specially adapted light traps for collecting free-living stages of gnathiid isopods are outlined. Sample results and possible modifications of the basic protocol for a variety of different sampling needs are presented and discussed.

For sampling in the central Philippines, the outlined trap design (Figure 1) was used. When 36 traps were set overnight (in one site), 1 to 1343 gnathiids per trap (275 &#177; 54) were collected. These included both fed and unfed juvenile stages (Figure 2; Table 1, 2). These results demonstrate the effectiveness of light traps for collecting gnathiid isopods under the study conditions. Figure 3 shows the placement o...

Watch this Scientific Journal Video about Collecting Marine Gnathiid Isopod Fish Parasites with Light Traps at JoVE.com

Collecting Marine Gnathiid Isopod Fish Parasites with Light Traps

We present a method to collect marine gnathiid isopod fish parasites using light traps placed at field sites via breath-hold diving or scuba diving.

A method to collect marine gnathiid isopod fish parasites with the use of light traps is presented. Gnathiid isopods are a major group of marine fish ...

Parasitic crustaceans are important factors in the ecology and life history of reef fishes. With the biomass and energy they removed from their hosts being considerable and influencing behavior, physiology, and survivorship. Gnathiid isopod crustaceans represent the most prominent group of fish parasites in tropical and subtropical reef systems, where they are both abundant and diverse and are the primary food item of cleaner fishes.Gnathiids have unusual life histories in which only the three juvenile stages feed on blood and body fluids of fishes, while in the adult stage, they no longer feed. Given that feeding requires only a brief association with the host, while each interfeeding interval last days, Gnathiids spend most of their life freeliving in the benthos. Given the many ways in which Gnathiids impact their hosts, there are compelling reasons to include them in ecological studies of coral reefs.However, their unique life history and the small number of researchers who study them creates a barrier to the development, implementation, and dissemination of reliable reproducible sampling methods to collect them for research. Light traps have long been used to collect small marine organisms at night. They take advantage of and are based on the fact that many nocturnally active organisms, including arthropods, are attracted to light.Traditionally, they have been used to collect planktonic organisms in the water column. However, the basic principles can be applied to collecting free swimming organisms that are active near the benthos. Here we present a light trapping method adapted for collecting free living stages of Gnathiid isopods near the ocean bottom in remote coral reef environments such as the Philippines.In collecting remote areas, these traps offer some advantages over other methods that have been developed for collecting these organisms. They're highly portable and durable and they require only three parts, which are easily obtainable and inexpensive. They're also negatively buoyant.Because they're dependent on light for attraction, they're only effective at night for collecting nocturnally active species. They also attract more than the target species, requiring sorting of the samples under a dissecting microscope to obtain the target organisms. Light traps.Construction.Light traps are best constructed from commercial PVC tubes originally designed for plumbing purposes. Use 20 to 25 centimeter diameter PVC cut to 30 to 40 centimeters in length. To both ends of the tube, add PVC caps with transparent acrylic funnels inserted in the center of the opening and glue in place with transparent epoxy glue.Ensure that one end of the tube has a screw on otherwise removable lid and that both ends are water tight when the trap is closed. Light source. Before deployment, an underwater torch, such as a Fantasea nano spotter 6023, should be turned on and placed in the tube facing the transparent funnel such that the light from the underwater torch illuminates the outside in front of one side of the tube.The light attracts a variety of small nocturnal organisms, including Gnathiids, and drives them to swim into the tube with the clear funnel. Once they've entered the tube, they're unable to escape due to the geometry of the light trap and the continued presence of a light source.Placement. Fill light traps with the light turned on and with sea water and secure both ends.Place traps on the sea floor in the sand or rubble next to coral heads or other complex structures known to attract fish. Focus the light cone inwards towards the areas where fish aggregate. In shallow water, the traps can be placed by breath hold diving.Deeper deployment requires scuba.Retrieval. Organisms will remain in the trap once the batteries of the lights have expired and the light is no longer illuminated. This provides flexibility in the time in which the traps are retrieved.Immediately prior to retrieving the trap, seal the openings of both the funnels on either end of the tube with a piece of clay or rubber stopper sealing, keeping all the seawater and the contained organisms inside.Transport. Once the traps have been retrieved from the bottom, they can be carried by divers to a boat or swam ashore. However, it's best to maintain traps as close as possible to ambient seawater temperature once they've been removed from the ocean.Transport them to the laboratory for processing as soon as possible since no gas or water exchange will take place once removed from the ocean. Laboratory processing. Storing and filtering samples.Once a light traps are removed from the ocean and brought back to the lab, empty their contents into buckets with fresh sea water. Add aeration to keep organisms alive until filtering. Filter the contents of the bucket by pouring through a funnel lined with 50 to 100 UM plankton mesh.Then empty the contents to a hundred milliliter container of fresh seawater. Use a pipette to draw from this small container to place aliquots of the sample into a Petri dish for microscopy. Repeat until the entire sample has been processed.Gnathiid isopod identification and rearing. Because light trap samples will attract multiple species of small invertebrates, samples must be carefully screened to identify and remove Gnathiids isopods. 10 to 20 times magnification is best for this task.Identifying Gnathiids at the family level does not require living specimens. However, adult Gnathiids, which are seldom caught in light traps, are needed for morphological species ID and for breeding. Thus, in cases where Gnathiids need to be kept alive of rearing, gently remove them with a pipette and place them in small plastic containers of fresh seawater.Representative results. The sampling in the central Philippines, the outline trap design was used when traps were set overnight. One to 1, 343 Gnathiids per trap were collected.These included both fed and unfed juvenile stages. These results clearly demonstrate the effectiveness of light traps for collecting Gnathiid isopods under the study conditions. This method for Gnathiid collection is effective and sufficiently flexible for a variety of field sites and scientific questions.For example, in a study by Artim et al in 2020 in the Caribbean, where they used light traps to quantify coral cover and fish biomass on Gnathiid abundance.Conclusion. While other techniques have been developed and used for collecting Gnathiids, this technique is particularly efficient. While the light traps described here attracts a wide range of small mobile invertebrates, the composition of this bycatch is highly variable.However, when set in habitat that includes rubble and fish, Gnathiids are caught consistently. Although this demonstration study focused on sites in the Philippines, traps of a similar design have also been used successfully for studies at sites in the Caribbean region and the Great Barrier Reef.

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Research

JoVE Journal

Biology

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.&#160;

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...