About those lancelets

[seabass]

So are you just curious, or are you planning on trying to keep one or more? You'll get more information searching the Internet than by asking here, as they are not sold in the aquarium trade. When you're searching, you'll notice a few threads where people found them in their tanks (as a hitchhiker). However, I haven't seen threads where people have kept them long term. Do some more research and post your findings in this thread.

[amphipod]

I was planning on winter vacation to Florida I go lancelet hunting, and was planning on trying them out with close to nature care, their homelands sand, everything. I'll continue research and post more info, and if I catch any detailed experience..

[amphipod]

I found this that a Japanese lab has used to culture lancelets. http://www.researchgate.net/publication/256449827_Stable_aquaculture_of_the_Japanese_lancelet_Branchiostoma_japonicum_for_7_years

[tibbsy07]

That paper is interesting, but it brings up a problem with lab work - sometimes the organisms we keep in the lab become domesticated, not like pets, but we select for the bacteria/viruses/fish/etc. that have continued living in our lab conditions and continually use them. Over time we allow for dominant phenotypes/genotypes that sometimes (not always, though) fail to mimic what is actually occurring in the wild. What that means is that they are the only ones with lancelets in captivity, and those are in lab conditions, in which those specific lancelets have adapted to survive - and my guess is that in order to be successful in your home tank with lancelets, you'll need to get specimens directly from them for the best chance. They probably lost a lot in the process, by the way.

[amphipod]

I downloaded the whole article on my laptop, not just the abstract. amazingly they started with wild Japanese lancelets and originally had many losses due to bacterial infections. Then they found deep sand beds were a necessity to keep them alive. Over the 7 years the article covered they only started the culturing method for lancelets allowing such a useful creature to potentially be used in more labs.

[amphipod]

I wonder how the best way to catch them by hand would be?

[charnelhouse]

Can you please post the text of the full article? I'm surprised it doesn't discuss how to harvest them by hand.

[amphipod]

How do I go about it, its in PDF format?

[amphipod]

When you open my link I pasted earlier click view for the abstract then click download from the top right corner. That's how I got the full article.

[amphipod]

This Saturday I'm heading to Florida for break, then I shall search for the lancelets.

[seabass]

You might need a shoreline fishing license: http://m.myfwc.com/license/recreational/saltwater-fishing/shoreline-faqs/ I believe that you can get one for free.

[amphipod]

Hecks yeah, I qualify as an exemption for age, and not hunting game species.

[charnelhouse]

How do I go about it, its in PDF format?

 

Do an OCR Text Recognition then you can just copy and paste each letter one at a time.

[amphipod]

Do an OCR Text Recognition then you can just copy and paste each letter one at a time.

Yeah, that's not happening.

[charnelhouse]

Yeah, that's not happening.

 

Please? I would think that with your dedication to the observation of the out of the ordinary a few hours of copy/paste would be easy.

[hypostatic]

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071599

[mechishark7]

So, not trying to be a jerk, and I know your the kid who likes to keep aiptasia...but with all of the amazing marine organisms that are steadily kept in this hobby, why on earth would you want to keep these in a tank?

[amphipod]

So, not trying to be a jerk, and I know your the kid who likes to keep aiptasia...but with all of the amazing marine organisms that are steadily kept in this hobby, why on earth would you want to keep these in a tank?

I have always had an immense fascination with them, their unusual habitshabits and lesser known biology have kept me interested always.

 

I have caught amphipods, isopods, polycheates, anoles, mangroves, an assasin bug, red algae, a rosy wolf snail, barnacles, and a foot long horseshoe crab, and still no lancelets. I was also bit by a wood stork i fed fries to.

[mrBananas]

Can you please post the text of the full article? I'm surprised it doesn't discuss how to harvest them by hand.

 

Do an OCR Text Recognition then you can just copy and paste each letter one at a time.

 

How do I go about it, its in PDF format?

 

If you follow the link Amphipod posted and in the upper right corner click "Full Text" then it brings up the PDF and an option at the top for Plain Text, which I've copy/pasted below.

 

_____________________________________________________________________________________

 

 

Stable Aquaculture of the Japanese Lancelet Branchiostoma japonicum for 7 Years KINYA YASUI1*, TAKESHI IGAWA2, TAKAO KAJI1, AND YASUHISA HENMI3 1Department of Biological Sciences, Graduate School of Science, Hiroshima University, Higashi‐Hiroshima, Hiroshima, Japan 2Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashi‐Hiroshima, Hiroshima, Japan 3Aitsu Marine Station, Center for Marine Environmental Studies, Kumamoto University, Kami‐Amakusa, Kumamoto, Japan For understanding chordate evolution, studies on vertebrates and urochordates have innovated on experimental techniques to obtain compelling data, whereas the remaining group, cepha- lochordates, has delayed research owing to inherent difficulties in working with this taxa. In 2008, a sequence dataset of Branchiostoma floridae genome and ESTs (expressed sequence tags) became available (Putnam et al., 2008), which resulted in largely improved gene cloning for lancelet species and design of geneticmanipulations.Additionally,tomakelanceletsconvenient for routine use in the laboratory, laboratory cultures have been performed at several institutions (Fuentes et al., 2004, 2007; Yasui et al., 2007; Zhang et al., 2007; Somorjai et al., 2008; Theodosiou et al., 2011). Of these, the species Branchialstoma lanceolatum has been successfully induced to spawn by thermal shock in captivity (Fuentes et al., 2004, 2007; Theodosiou et al., 2011) and also maintained in complete artificial seawater at inland institutions ABSTRACT Despite advances in the study on animal evolution in the last two decades, paucity of experimental data on cephalochordates comparable to those on the other chordates hinders an integrative understanding of chordate evolutionary history. To obtain lancelet data under well‐controlled experiments, laboratory cultures of lancelets have been performed at several institutions. In a mass culture started in 2005, we have obtained up to three consecutive generations of Branchiostoma japonicum. Using sand substratum, survival rates of laboratory lancelets until maturation have improvedtogreaterthan30%,muchhigherthancomparedtopreviously,andforadultstheannual average survival rate was 82.3%. The high survival rate allows maintaining animals at least 6 years and potentially longer. Water temperatures lower than 23°C obviously reduced the frequency of spawningevenaftertheonsetofspawningperiod, and1–2days afterchangingthetemperatureat 25°C animals became spawned well. We also observed obvious sex reversal from male to female in individuals that had been cultured for 3 years or more. Our continuous culture has provided sufficientmaterialsfor vitalexperiments onearlydevelopment andforstudying metamorphosis, as well as for the conservation of wild populations. The subculture of successive laboratory generations will provide a valuable resource for genetic studies. J. Exp. Zool. (Mol. Dev. Evol.) 320B:538–547, 2013.©2013 Wiley Periodicals, Inc. How to cite this article: Yasui K, Igawa T, Kaji T, Henmi Y. 2013. Stable aquaculture of the Japanese lancelet Branchiostoma japonicum for 7 years. J. Exp. Zool. (Mol. Dev. Evol.) 320B:538–547. J. Exp. Zool. (Mol. Dev. Evol.) 320B:538–547, 2013 Grant sponsor: RIKEN Center for Developmental Biology. ?Correspondence to: Kinya Yasui, Department of Biological Sciences, Graduate School of Science, Hiroshima University, 1‐3‐1 Kagamiyama, Higashi‐Hiroshima, Hiroshima 739‐8526, Japan. E‐mail: furaha@sci.hiroshima-u.ac.jp Received 28 March 2013; Revised 3 August 2013; Accepted 6 August 2013 DOI: 10.1002/jez.b.22540 Published online 4 September 2013 in Wiley Online Library (wileyonlinelibrary.com). RESEARCH ARTICLE ©2013 WILEY PERIODICALS, INC.

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(Theodosiou et al., 2011). For the sake of genetic and develop- mental studies, a culture system capable to maintain individual adults during at least 5 months has also been developed (Somorjai et al., 2008). These challenges provide a variety of methods applicable to a wide range of requirements. Since 2005, we have maintained the Japanese lancelet Branchiostoma japonicum (former B. belcheri; Zhang et al., 2006) in a mass‐scale laboratory culture in order to get sufficient developmental samples for a variety of experiments and to establish a closed laboratory colony with minimum labor (Yasui et al., 2007). In this culture system, we have obtained gametes and progeny every year, and some of these progeny have been maintained for subcultures. Although sand substratum was not initially used to make monitoring easy and to save labor, we have noticed that sand is important for healthy development and protection against bacterial infections. We have thus set sand substratum in adult tanks, and offspring are transferred into adult tanks when they become juveniles larger than 1cm. The culture with sand substratum drastically improved the survival rate and saved labor. Food was simplified to a single species of alga, Chaetoceros gracilis, and it has turned out that feeding with this species is sufficient for sexual maturation and annually repetitive gonad development. In this culture system, first (F1) and second (F2) laboratory generations became sexually matured and spawned every year. Third laboratory generation individuals (F3) bred from F2 are now being maintained in adult tanks. These successive generations have gradually reduced the mean number of alleles and could be established as an inbred line in the future. As the colony supplies sufficient gametes and embryos, though limited to the breeding season, they can be utilized for many different experiments and the conservation of wild populations. MATERIALS AND METHODS Animals The founders of the maintained colony were adult lancelets collected from the Ariake Sea, Kumamoto, Japan, prior to the breeding season every year from 2005 to 2012, with the colony having been maintained in separate 2.5‐L adult tanks. Sexually mature individuals were separated by sex that was determined by gonadsunder microscopic inspection. Progeny was obtained from eggslaidbyafemaleorseveralfemales,whichwerefertilizedwith sperm from a male or several males. Culture of Adults and Juveniles Animals collected from the wild and of offspring that grew up to juveniles larger than 1cm were cultured in the Hydense System (Aqua Co. Ltd, Tokyo, Japan) with slight modifications from our previous report (Yasui et al., 2007). Although we originally did not set sand in the tanks, sand from the natural habitat sieved with 3‐mm mesh was set at a 2‐ to 3‐cm depth in the tanks. Sand substratum was removed before spawning started in June and set again with sand that was washed with tap water and well dried by sunlightaftertheendofthebreedingseasoninAugust.Inaddition to sand removal, sand was cleaned once or twice in a year by washing thoroughly with seawater, and tanks were also washed with tap water at the same time. Adult animals were usually maintained at 20–30 individuals per 2.5‐L tank. The culture room was air‐conditioned at 24°C year‐round under natural day/night cycle through the window. Changes of seawater temperature were recorded with a data logging thermometer (HOBO Pendant Temperature/Alarm Data Logger 64K‐UA‐001‐64, Onset Comput- er Corp., Bourne, MA) set in an adult tank in the system. Daily Care Running seawater was pumped from a depth of 421cm from the half‐tide level (165cm below extreme low tide line) at the Marine Station of Kumamoto University and stored in an outdoor 50‐ton seawater tank. Running seawater was supplied directly from the tankwitha seawater supply line at9:00 and was stoppedat 16:30. Feeding with 60‐mL culture medium of C. gracilis (9–13?106 cells/mL) was done soon after stopping the running of the seawater. Preparation for Collecting Gametes and Embryos Sand substratum was removed 1–2 weeks prior to starting collection of gametes and embryos. Removed sand was washed with tap water thoroughly and dried by sunlight for reuse. Tanks were cleaned with tap water and rinsed with seawater, and then animals were returned into cleaned tanks without sand. Before starting seawater supply every morning, each female and male/ female mixed tank was checked to examine if there were embryos in seawater or females that had emptied gonads. Collection of Gametes and Embryos During the period of gamete and embryo collection, running seawaterwasstoppedat15:00andlanceletsfedwith100mLofthe algal culture medium. At 18:00, in order to wait for spawning, seawater in each tank was drained and left at a depth of approximately 2cm with aeration also stopped. Spawning was checkedeveryhourfrom21:00,andeggsorsperminthetankwere collected with seawater, separating adult animals with a mesh. Seawater in a container that contained eggs was changed with filtered seawater as much as possible and then eggs were fertilized with fresh sperm. After finishing spawning checks between 24:00 and 1:00, the tank was filled with seawater and aeration restarted. Culture of Progeny Fertilized eggs from one or several females (depending on the numberofeggs)wererearedin300‐mLplasticcontainersuntilthe neurula stage of approximately 16hpf and then transferred and maintained in 33‐L glass tank without sand substratum at 24°C as reported previously (Yasui et al., 2007). Daily feeding was started at36hpfwith100mLofthesameC.gracilisculturemediumasfor STABLE CULTURE OF JAPANESE LANCELET539 J. Exp. Zool. (Mol. Dev. Evol.)

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adults for the first 2–3 days, and then the volume of food was increased to 200mL/day in the first month, 300mL/day for next 2 months, and 400mL/day until transfer into adult tanks. Seawater changes and tank cleaning were done as previously reported (Yasui et al., 2007). When metamorphosed juveniles reached more than 1cm at the end of November or in December, theyweretransferredintoadulttankswithsandsubstratumsieved with 2‐mm mesh. The number of individuals in a tank was approximately 500 individuals at the beginning and reduced to 50–100 individuals by sexual maturation, at which they had reached approximately 2.5cm in length. Measurement of Body Size of Progeny Two populations of F1 that bred on July 25, 2006 and on August 19, 2007 were measured monthly. The F1 bred in 2006 were first measured as a random sampling of 20 individuals from theglasstank.Themeasuringmethodwasthenchangedtousethe same 24 individuals to which eight individuals were added later, all of which were maintained in a separate adult tank. The F1 bred in2007weremeasuredusingthesame43individualsinaseparate adult tank. Change of Seawater Temperature for Controlling Spawning To know if the spawning of B. japonicum could be controlled, the roomtemperaturewasregulatedbyairconditioner.Initiallytheair conditioner was set at 20°C on June 23, 2012 and changed to 25°C on July 3. For the following 3 weeks, after finishing spawning checks around 24:00 to 1:00 on July 7/8 and 14/15, the air conditioner was lowered from 25 to 20°C. This low temperature was kept for 41–42hr and them returned to 25°C. Seawater temperaturesinanadulttankwererecordedduringthistreatment. Genotyping of Microsatellite Loci and Genetic Analyses for Laboratory Generations DNA was extracted individually from 20 individuals from the founder and F3 populations and from 10 individuals from F1 and F2 by using NucleoSpin Tissue (MACHEREY‐NAGEL, Düren, Germany). These individuals were genotyped using 10 microsat- ellite loci previously reported; Branb3, Branb5, Branb13, and Branb17(Liuetal., 2009),as wellas1113‐4H,0106‐2D,0110‐9C, 0112‐13A, Branb‐3, and Branb‐5 (Li et al., 2011). PCR amplification was performed based on M13 tag method (Schuelke, 2000), adding an M13(‐21) sequence at the 50end of one of the primer pairs and M13(‐21) primers labeled with fluorescent dye (FAM, HEX, NED, or PET) in each reaction. Ampliconswere mixedwithGeneScanLIZ500(LifeTechnologies, Carlsbad, CA) as an internal size standard and electrophoresed by using ABI3130xl (Life Technologies). Fragment sizes and genotypes of the amplicons were then determined by using GeneMapper 4.0 (Life Technologies). Based on the departure from Hardy–Weinberg equilibrium (HWE) calculated with Genepop version 4.0 (Rousset, 2008), loci showing significant excess or deficiency of HWE in all generations were omitted from the following analyses. We finally calculated the number of alleles (Na), observed heterozygosity (HO), expected heterozygosity (HE), and Fixation index (F) for each generation by using GenAlEx 6.5 (Peakall and Smouse, 2012). Phylogenetic Analysis To clarify the taxonomic status of our laboratory colony, five individuals of the founder population, from which DNA was extracted for genotyping, were used for molecular phylogenetic analyses based on mitochondrial 12S rRNA gene. PCR amplifica- tion ofa DNA fragmentincluding complete 12S rDNA, Pro‐, Phe‐, and Val‐tDNA, as well as partial 16S rDNA was conducted accordingtotheprotocolwiththeprimerset(Amph‐Thr‐F/Amph‐ 16S‐R) described in Xu et al. (2005). Amplicons were sequenced from both directions to obtain reliable consensuses. The resultant sequence for each animal was deposited in EMBL/GenBank/DDBJ database (Ariake‐1, AB820695; Ariake‐2, AB820696; Ariake‐3, AB820697; Ariake‐4, AB820698; Ariake‐5, AB820699). For molecular phylogenetic analyses, we used only the complete sequences of 12S rDNA as the gene has the largest number of registrations of genus Branchiostoma in the database. We aligned the haplotype sequences with the 31 haplotypes of four species of genus Branchiosotma (29 haplotypes registered as B. belcheri, B. belcheritsingtauense,andB.japonicumfromJapanandChinaand each single haplotype of B. floridae and B. lancelatum) using MUSCLE (Edgar, 2004). After alignment, ambiguities and gaps were excluded from phylogenetic analysis using Gblocks version 0.91b (Castresana, 2000) with default setting. Phylogenetic trees were constructed both with Neighbor‐Joining (NJ) and Maxi- mum‐Likelihood (ML) methods seeking a suitable substitution model for those analyses under MEGA 5 (Tamura et al., 2011) with B. floridae and B. lancelatum as the outgroup. For ML inference, an NJ tree was used as the initial tree and Nearest‐Neighbor‐ Interchange (NNI) were used for tree search method. Support for both resultant trees were evaluated with bootstrap proportions of 1,000 pseudoreplicates. RESULTS Taxonomic Status of Laboratory Colony Although the species of genus Branchiostma in Japan was previously assigned to Branchiostoma belcheri (Grey, 1847), it has been proposed to reclassify Japanese specimens to Branchistoma japonicum (Willey, 1897) based on the two distinct species in Xiamen waters in China (Zhang et al., 2006). As variation in the sequences of mitochondrial 12S rDNAcan distinguish between B. japonicum and B. belcheri (Xu et al., 2005), we performed sequence alignment of this gene. For the five founder animals collected from the field (Ariake Sea), each animal had a unique haplotype of 12S rDNA ranging from 2 to 7 nucleotide differences between them. Trees constructed both with ML (HKYþI model; J. Exp. Zool. (Mol. Dev. Evol.) 540YASUI ET AL.

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Hasegawa et al., '85) and NJ (TN93 model; Tamura and Nei, '93) methods based on 805 nucleotide sites showed the same tree topology. Thefivefounderanimals were grouped intoa cladewith specimens assigned to B. japonicum and B. belcheri tsingtauense fromXiamenandQindao,China,aswellasfromotherlocationsin the Japanese coastal waters (all currently assigned to B. belcheri) with 100% bootstrap support (Supplementary Material, Fig. S1), whereas other specimens assigned to B. belcheri from China clustered in another distinct clade. As the genetic distance of the haplotypes between the clade including B. japonicum, B. belcheri tsingtauense, and all specimens from Japan and the clade including specimens assigned to B. belcheri from China was comparable to that between B. lanceolatum and B. floridae, we identified our laboratory colony as B. japonicum. Outline of 7‐Year Culture The culture was started in October 2005 without sand substratum (Yasui et al., 2007). It turned out, however, that long‐term culture withoutsandsubstratumcausedthetankstorapidlybecomedirty, which then caused bacterial infections of animals in 2.5‐L adult tanks,especiallyinsummerseason.Thecultureswithoutsandalso induced malformations of the notochord and oral structures in progeny when maintained in 33‐L glass tanks (Yasui et al., 2007). We have utilized sand substratum since November 2006. To maintain the laboratory colony, we collected 200–500 wild lanceletsinthelatterhalfofJunesince2005andstartedtoculture lancelets in the system without sand substratum for checking spawning (Fig. 1). Temperature of seawater in an adult tank set in the system from January 2010 to December 2012 is shown in Figure 2. Although we set the air conditioner at 24°C, the temperature of running seawater in the culture system could not be controlled precisely as seawater was supplied from the outdoor seawater tank. The seawater temperature fluctuated annually between 11 and 28°C, and larger daily changes averaged approximately 6°C were observed generally in winter but also in the summer of 2012. Spawning in the system started from mid‐ June when water temperature rose above 23°C and continued to the end of July or late August, depending on the year. A population of F1 bred on July 25, 2006 was subcultured without sand substratum until the end of October 2007 and then transferredintoadulttankswithsand.ThisF1generationbeganto develop gonads at the end of October 2007 and spawned in mid‐ Figure 1. Culture and spawning records of wild and laboratory generations. (A) Field collection, spawning, and flowchart of laboratory generations from 2006 to 2012. Black arrows and dark yellow arrowheads denote field collection and spawning observed, respectively. Color lines indicate history of laboratory generations. The record of 2010 is omitted as there were no progeny maintained. ( Larvae of third generation 4 weeks after fertilization. J. Exp. Zool. (Mol. Dev. Evol.) STABLE CULTURE OF JAPANESE LANCELET 541

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June 2008. From their spawn, a batch bred on June 15 was subcultured as an F2 generation. The animals of the F2 were transferred into adult tanks with sand substratum at the end of November 2008 when they were juveniles of 1cm or more in length. As the F2 generation spawned in June 2010 and 2011 like their parental generation, we tried to culture their offspring. The larvae, however, did not grow normally and finally died in the 33‐ L glass tank in both years. The F2 again spawned in June and July 2012. From their spawn, we have been subculturing a new F3 population (Fig. 1). This F3 generation was transferred into adult tanks with sand substratum on December 25, 2012. The oldest animals have been maintained since they were collected from the field in 2006 and have been living more than 6 years. The F1 bred in 2006 has also survived for 6 years. As severaltenthousandsoflarvaesurvivedtojuvenilesoflargerthan 1cm in length, we released juveniles from F1 generations into the original habitat in 2008 (approximately 14,000 individuals) and 2012 (approximately 8,000 individuals). Survival Rate We checked survival rates from 2010 to 2012 for animals collected from the wild habitat and the laboratory generations (Table 1). When the animals were grouped according to collect- ing year, the lowest survival rate was 47.6% and the highest was 89.7% during these 2 years. There was no tendency of animals that were maintained longer period to have a lower survival rate. As the survival rates varied annually in the same year group, rates seemed to be affected mainly by temporal culture conditions. The survival rate of progeny was 2% when cultured without sand substratum (Yasui et al., 2007). We have modified our culture methodtotransferjuvenilesintoadulttankswithsandsubstratum when they became larger than 1cm at 4–6 months after fertilization. This modification has surprisingly improved the survivalrate.Asitwastechnicallydifficulttoestimatethenumber of fertilized eggs or early embryos, we first estimated the number of larvae 1–2 weeks after fertilization. The number of individuals reduced to 35–70% of the first estimate during the first 6 months culture in the glass tank, and when transferred into adult tanks, the estimated number of juveniles was ca. 14,000 and ca. 20,000 for the F1 and the F2 bred in 2008, respectively, and ca. 8,000 and ca. 25,000 for the F1 and the F3 bred in 2012, respectively. The survival rate of the laboratory generations after transfer into the adultculturesystemfrom2010to2012washigherthan85%inthe twoF1populationsbredin2006and2007(Table1).Asubgroupof the F1 bred in 2007 had a long‐term survival rate of 39.5% from 2007 to 2012. This agrees well with the mean annual survival rate of 82.3%. Accordingly, we can obtain sufficient number of sexually matured progeny for multiple laboratory demands of studies and subculture of laboratory generation, as well as for releasing them into the field. Figure 2. Change of seawater temperature in culture system from 2010 to 2012. Upper and lower lines denote highest and lowest temperatures each day, respectively. Table 1. Annual survival rates from 2010 to 2012. Group Collected in 2006 2007 2008 2009 2010 F1 of 2006 F1 of 2007 2010–2011, % (no. ind.) 2011–2012, % (no. ind.) 2010–2012, % 100.0 (9/9) 61.9 (52/84) 49.8 (114/229) 96.4 (134/139) 55.5 (5/9) 84.6 (44/52) 95.6 (109/114) 73.4 (99/134) 76.0 (165/217)a 86.8 (269/310) 91.8 (279/304) 55.5 52.4 47.6 71.2 — 86.8 89.7 100.0 (310/310) 97.7 (304/311) Mean annual survival rate: 82.3%. aFemale only. J. Exp. Zool. (Mol. Dev. Evol.) 542 YASUI ET AL.

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Growth of Laboratory Generations We measured the body length of the two F1 populations bred in 2006 and 2007 (Fig. 3). The F1 bred in 2006 took 6 months and the 2007F1took3.5monthsuntiltheybecamelargerthan1cmlength at 24°C in the same type of glass tanks. The F1 bred in 2007 constantly grew in the first year at the growth rate of 1.5mm/ month and reached approximately 3cm by November 2008. However, thereafter their growth was very slow, and none of them exceeded 4cm in length. The 2006 F1 caught up the size of those bred in 2007, taking 2.5 years. The F1 bred in 2006 showed malformations (Yasui et al., 2007), and their growth was apparently affected by these malformations. However, long‐ term observation has shown an interesting feature. The 2006 F1 stopped growth from February to spawning season and then grew again (Fig. 3). In our culture system, animals usually started to developgonadsinOctoberandreleasedgametesfrommid‐Juneto August. The stagnation of growth seems to synchronize to the development of gonads. In the 2007 F1, animals reached more than 2cm in April 2008 and developed gonads irregularly in this month. Gonads were not released in the breeding season in the same year and degenerated just before the normal onset of gonad developmentinOctober.Duringthisirregulargonaddevelopment, the 2007 F1 did not show a stagnation of growth, however. Sex Reversal Sex reversal from female to male was reported in a Chinese population of B. japonicum (former B. belcheri tsingtauense) (Zhang et al., 2001). In our system, in which adult animals were separately maintained by sex, another case of sex reversal was observed in2012, which was the first timein our culture during its 7‐year period (Table 2). While the previous sex reversal reported was from female to male, the present sex reversal was only from male to female. The highest reversal rate of 50% was seen in the males of the 2006 F1. Males collected from the field in 2007 and those of the 2006 F1 also produced hermaphrodite individuals (Fig. 4). As reported previously (Orton, '14; Riddell, '22; Chin, '41; Yamaguchi and Henmi, 2003), hermaphrodite individuals devel- oped several ovary sacs in the majority of testicular sacs in the gonad. Some females developed whitish gonads similar to male ones, but the identification of their sex was not difficult under microscopic inspection, whereas small males were rarely misidentified as females. This may raise the issue of possible mis‐sorting of males and the placement of them into female tanks in the preceding year. However, this inaccurate identification did not affect the present sex determination. In the tanks in which the sex reversal occurred, all individuals showed easily identifiable Figure 3. Growth curves of two first laboratory generations measured by focal sampling. Progeny bred in 2006 magenta and that in 2007 green. Bars indicate largest and smallest individuals. Numerals with triangle or rectangle: 1, fertilization in 2006; 2, fertilization in 2007; 3, transfer into adult tank; 4, gonad development; 5, transfer into adult tank; 6, gonad degradation; 7, gonad development; 8, spawning; 9, gonad development. J. Exp. Zool. (Mol. Dev. Evol.) STABLE CULTURE OF JAPANESE LANCELET 543

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sexual characters such as color and outlines of oocytes. There was nomalefoundinthefemaletanks.Althoughwecheckedthesexof animals in each tank when preparing for collecting gametes and embryos each year, we did not recognize sex reversal until 2012. Aswedidnotchangetheculturingmethodduringtheseyears,and though the system was moved from the Marine Laboratory of Hiroshima University to the Marine Station of Kumamoto University in 2010, it remains unknown what caused the sex reversal from male to female. Preliminary Observation of Change in Spawning Frequency by Temperature Control In order to suppress spawning until the start of collection of research materials and on weekends, we changed the room temperature from 25 to 20°C from June 23 to July 3 and for 41– 42hr from midnight on July 7 and 14 in 2012, as animals in our system start spawning when seawater temperature became higher than 23°C. Temperature of seawater in the culture tanks during this “control” of the room temperature is shown in Figure 5. We performed three repeats of the temperature control and observed that there were no or few spawning animals during the low temperature setting, and that the number of spawning animals increased 1 or 2 days after re‐setting the air conditioner at 25°C (Fig. 5). As we were unable to predict on which day animals spawned before 2012, the three repeats of the high frequency of spawning during the high temperature setting improved our methodology. Continuous spawning as observed during the high temperaturesettingin2012wasthefirstcaseinour7‐yearculture and thus suggests a possible relationship between temperature changeandspawningofB.japonicum,similartothermalstimulus inB.lanceolatum(Fuentesetal.,2004).Weneedtorepeatthesame culture conditions or examine more precisely with control treatments to verify if the present treatment functions as a stimulus for spawning. Table 2. Sex reversal observed in 2012. Group Collected in 2007 2008 2009 2010 2011 F1 of 2006 F1 of 2007 No. of ind. in < tanks in 2012a No. of < to , No. of , to < Sex reversal rate (%) No. of hermaphrodites 25 35 37 143 50 88 126 4 6 6 0 0 0 0 0 0 0 16.0 17.1 16.2 32.9 10.0 50.0 25.4 1 0 0 0 0 1 0 47 5 44 32 aNumber of surviving individuals in 2012 that were identified as male in 2011. Figure 4. Gonads of hermaphrodite individuals. Arrowheads indicate gonadal sacs containing oocytes. Figure5. Numberoftanksinwhichanimal(s)spawnedandchange of seawater temperature during controlled room temperature setting at 20 and 25°C. J. Exp. Zool. (Mol. Dev. Evol.) 544YASUI ET AL.

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[amphipod]

Today I caught hermit crabs, some assorted small crabs, several polycheates, most which have skin that has an unusual Velcro like sticky feel to skin, weird. Also a nudibranch, tube worms, comb jellyfish, sponges, weird planktonic organisms, and a dead puffer fish.

 

What would you feed a comb jellyfish in captivity?

[cindyp]

I was also bit by a wood stork i fed fries to.

 

[amphipod]

Lol it was hilarious, there were like 8 others who got all the other fries and he was just standing in the sidewalk staring, so I offered the fry directly to him and he got quite excited and bit my finger with the fry.

[seabass]

What would you feed a comb jellyfish in captivity?

See: http://www.nano-reef.com/topic/355405-comb-jellyfish/#entry4914327

[amphipod]

Comb jellyfish are weird, I have saved around 30 today from fishermen who dumped them on the pier from mullet netting. I felt sorry for how helpless they were.

[amphipod]

I hate having to leave the campground. It was so fun at the water.