© 2013 Nature America, Inc. All rights reserved. the globe. Here we present protocols for collecting collecting for protocols present we Here globe. the reef ecosystems and threatened by extinction in many locales across corals, which are crucial to the function and persistence diverseof an outgroup to inform studies on the closely related reef-building and are based. As an anthozoan cnidarian, it also serves as mals’ upon which most studies of development, physiology so-called the ani of ‘higher independent is that animal from deuterostomes— protostomes of divergence the before years million 50–75 some —which emerged as an independent evolutionary lineage (endo ( gastrodermis dermal) inner the and epidermis (ectodermal) outer the layers, animal that lacks true organs, and it is composed of only two tissue in whole ( visualize the internal toanatomy (and possible labeling it of RNAs making and proteins)transparent, mostly is anemone the cycle, mature adult under ideal culture conditions) generation time is short (~50 d from the fertilized egg to a sexually its and polyp) juvenile a to egg fertilized the from d (5–7 rapid is of development the stage, larval planktonic a with stocks clonal living of development the facilitates that property column,a body the through bisected when regeneration bidirectional complete undergo rapidly and shifts temperature and changes light feedings, regular of combination a through synchronized be can spawning of under artificial culture conditions sexual reproduction, undergoes larly forms as twoas well distinct laboratory the inexpensivein to culture and simple and laboratory the in studies field. It the is also in amenable to organismal, studies cellular, environmental molecular and and genomics ecological to amenable and natural range of anthropogenic environmental variation. It is anemone sea ronmental change. Its starlet geographic distribution spans a tremendous estuarine small, ( the respects, many In I specialized equipment. stock consisting of hundreds of genetically identical anemones. roughly weekly schedule. By inducing regeneration roughly every 2 weeks, in less than 6 months, one can establish a clonal it is possible to establish a laboratory colony that will be reliable in generating hundreds or thousands of fertilized eggs on a stocks. In less than 1 h at a suitable field site, a researcher on foot can collect hundreds of individual anemones. In a few months, how to collect system for field and laboratory studies of development, evolution, genomics, molecular biology and toxicology. Here we describe O Published online 11 April 2013; Biology,of Department Boston University, Boston, Massachusetts, USA. Correspondence should be addressed to J.R.F. ( Derek J Stefanik, Lauren E Friedman & John R Finnerty vectensis regeneration of the starlet , Collecting, rearing, spawning and inducing 916 ing clonal stocks by inducing regeneration. maintaining anemones in the lab, obtaining embryos and develop N. vectensis N. NTRO protocol ver the past 20 years, the ,

| VOL.8 NO.5VOL.8 D UCT ) is an ideal model system for this era of rapid envi rapid of era this for system model ideal an is ) I ON Nematostella | 2013 Figs. 1 | Nematostella natureprotocols Fig. and 1, doi:10.1038/npro 2 . 2 N. vectensis N. , , culture it through its entire sexual life cycle and induce regeneration for the production of clonal 2 ). As a representative of the phylum the of representative a ).As ). Morphologically, it is a very simple provides a perspective on early 9 . For a marine invertebrate marine a For . 3 t.2013.044 ( Fig. 6 . Throughout its life 1 7, ) is easy to collect collect to easy is ) 8 . It will reliably reliably will It . Nematostella Nematostella vectensis Nematostella Nematostella 4 .It regu 2,4– 6 . Its - - - - - ,

T hese hese results can be achieved very inexpensively and without A simpler and more efficient method, which is described here, is here,is described is which method, efficient more and simpler A disturbed. when pharynx the into tentacles their retract they and cult to reco diffi are anemones collector, inexperienced the for addition, In for ceps or a pipette transfer wide-bore using screen the from one-by-one anemones the pick and from suitable habitats estuarine over a fine screen mesh (~1 mm along the Boston neighborhoods waterfront. anemone at multiple estuarine habitats in heavily developed urban habitats that are degraded by human activity; we have collected this (e.g., in the range of 0.03–0.10 cm s depths at occurs it abundance; its habitats, rine flowand todepth of appear keybe determinants pools marsh salt to especially habitats, estuarine lished observations), but its occurrence appears to be restricted to (from values pH and p.p.t.) thousand, ( temperatures of to and coast Pacific England the to introduced been have to appears it America; North of coast Atlantic the is range native its that cates ( to Washington) England tencoast and of sites southern the along nine sites along the Pacific Louisiana), to coast of Scotia NorthNova (from America (fromAmerica North California of coasts Gulf to recover greater genetic diversity.functional the abundance of youbecause mayimportant be interested in the factors that affect is This habitats. widespread geographically or variable mentally through a vendor, is that you can choose to sample from environ them purchasing or colleague a from them acquiring than rather yourself, animals the collecting of advantage key A England. and easy to from organisms collect in Northcoastal estuaries America tors or sporadically from a handful of commercial vendors, it is also Although Animal collection Supplementary Data One published collection method is to sift the soft sediment sediment soft the sift to is method collection published One Nematostella , , a small estuarine animal, has emerged as a powerful model 1 0 Nematostella . g Nematostella nize nize on the screen, as they are transparent, practically has been reported at 40 sites along the Atlantic and Nematostella

−

t 2 °C) 28 to 1 and ref. can be obtained from individual investiga can tolerate an extremely wide range range wide extremely an tolerate can [email protected] 3 in the wild, or you may be seeking ). Population genetic analysis indi

<1.0 m, where flow rates are low are rates flow where <1.0m, 11–1 ).

− 1 0

3 1 . This approach is laborious. , salinities (2–52 parts per per parts (2–52 salinities , ). Nematostella

<7 to <7

>9; J.R.F.,unpub >9; 1 4 .estua Within is tolerant of

2 ) ------

© 2013 Nature America, Inc. All rights reserved. and eliminates the seasonality of and spawning eliminates the of seasonality a period of laboratory in conditioning difficult, actually be increases to the proven frequency has marine many in spawning induce to required conditions natural the replicating although Indeed, culture. laboratory in maturity in Uhlinger and Hand of work pioneering The genetics. studies of their physiology, functional development, conducting to impediment cell major biology a and been molecular has oratory in The and maintaining difficulty in spawning cnidarians the lab Animal culture and spawning tentacles extended. have their they when recognize to easy are anemones the because a higher yield of anemones from the same area of bottom sediment posture feeding a assume and substrate fromthe bodies their extend they ment to settle and retrieve the anemones with a transfer pipette as to collect the sediment into shallow trays, allow the disturbed sedi the foot or physa. Adult polyps typically have 10–16 tentacles. into three main regions: the head or capitulum; the column or scapus and individuals can reach several centimeters in length. The body can be divided typically ~1 cm long from mouth to tip of the physa, but in culture, well-fed Figure 1 c a Nematostella 1.0 mm 0.1 mm 1 mm

1 | 5 Adult polyp of . This procedure is less time consuming, and it results in being the first sea anemone to be raised to sexual sexual to raised be to anemone sea first the being Gastrovascular cavity Nematostella vectensis b Apical Mesenteries 0.1 mm tuf t Pharynx Gastrovascular Mesentery . Field-caught animals are cavity 4,16,1 Mouth Nematostella Tentacle 7 Mesentery . Capitulum or Pharynx head 4,1 Physa or Scapus or colum foot 6 resulted s case, ’s n * * - -

oral-aboral axis. The gametes also develop within the mesenteries. of longitudinal muscle fibers that are used to shorten the animal along its will eventually be eight mesenteries, and these structures will bear bundles tissue layers, as well as the pharynx and the first four mesenteries. There plane of this image reveals the division between endodermal and ectodermal time of settlement, the juvenile anemone has four tentacles. The deep focal to the mouth. ( At this stage, the larva is ~250 active swimmer, and it moves primarily in the direction of the apical tuft. prominent grouping of long cilia known as the apical tuft. The planula is an The asterisk indicates the location of the mouth. Opposite the mouth is a mesenteries that span the gap between the pharynx and the body wall. and ectodermal tissue layers, as well as the developing pharynx and the The deep focal plane of this image reveals the division between endodermal matrix is ~200 spawned egg mass. Each individual egg suspended in the gelatinous Figure 2 pared with the timing of mussel feeding water changes com of timing the correlated with morebe tightly separated by 24, 48 or 72 h, spawning in cue than feeding. When the mussel feeding and water changes were as food on the day after the water was changed and mussels were provided occurring events spawning the of 64% with week, per times 0.72 on the day after a water change and mussel feeding. Males spawned occurred events spawning these of 75% week,and per times 0.80 of average an spawned females regimen, this Under changed. is anemones are fed mussel ovary ( other anemones every day for 6 d ( protocol for spawning conditions that can be remedied by water changes. Their published inhibited by anoxia and possibly by high levels ammonia—two of it increases the concentration of dissolved oxygen and possibly possibly and oxygen dissolved of concentration the increases it water the Changing eggs. decreases the concentration metabolic waste of products, fewer whereas and fewer produced starved females the time, over However, food. without months several of course the over even alone, changes water of use the through that showed sistently on a 7-d schedule can begin to show diminished fecundity. (14 d) because, in our experience, individuals that are spawned con a with recent study bright light, increased temperature and feeding ( bines all four previously identified spawning cues—water changes, under at light 18 bright °C and monitored for spawning 15 every min. maintained are anemones the treatment, this Following 13 h exposure of to and light bright elevated temperature (25 °C). water change stimulus from Hand and Uhlinger feeding daily with d 10–14 for conditions dark °C), (18 cool in anemones rearing temperature ( elevated and light bright both to exposure simultaneous a under achieved fecundity highest the with spawning, induce also could Technauand pools tidal via inundation or rainfall. marsh salt into water of infusion the mimic might that manner a in spawning induce to serve could which of salinity, all the alters ad n Uhlinger and Hand Two findings suggest that water changes are a stronger spawning Here we describe a protocol for spawning Subsequently to the work of Hand and Uhlinger 4

| . Developmental stages of µ 8 Nematostella Fig. 3 c demonstrated that changing light and temperature temperature and light changing that demonstrated m m in diameter. ( ) ) Juvenile anemone 10 d after fertilization. Typically, at the b 7 7 . At the end of this conditioning period, the the period, conditioning this of end the At . ).An updated version of this protocol prescribes , we prescribe a conditioning longer of period Nematostella 4 demonstrated that spawning is clearly clearly is spawning that demonstrated natureprotocols can be entrained into spawning cycles cycles spawning into entrained be can µ b m m in length from the base of the apical tuft ) ) Planula larva 3 d after fertilization. Nematostella Nematostella vectensis Mytilus californicus Fig. 3 prescribes feeding feeding prescribes Nematostella 1 a 6 . In addition, Uhlinger ). On the day,eighth the

| Nematostella VOL.8 NO.5VOL.8 Fig. 3 4 is combined with protocol 4 . . ( , Fritzenwanker ) and the water a c was found to ) ) Freshly ). Consistent | Artemia 2013 that com

|

917 to

1 - - - 6

© 2013 Nature America, Inc. All rights reserved. • • EQUIPMENT • • • • • • REAGENTS M prepared on,be so you must and and oxygen, levels pH, nitrogen dissolved salinity, temperature, including variables, ronmental by separated differ dense in stands of marsh key grass—that envi pools small numerous with habitat marsh high a water—e.g., of bodies many semi-isolated small use a should site field harboring you tolerances, environmental different show that animals lect of distribution and abundance the affect that factors identify to wish you if example, For answer. to wish you questions the to tailored are they that so protocols culturing and sampling your design carefully should you culture, oratory collecting Before design Experimental benchtop. meter laboratory of N.vectensis of colony reproducing to sexually self-sustaining, a develop possible rapidly is it sex, each of individuals 7-d schedule. Beginning with a few founder tive spawning than placing one cohort on a produc consistently more in result can it d, 7 by staggered and schedule 14-d a on placed are animals of cohorts two Thus,if typically release eggs and sperm. treatment, the large majority of animals will the conclusion of the elevated temperature placed in bright light. Within a few hours of for a period of ~12–15 h and the animals are conditioning period, the temperature is elevated weekly water changes. At the conclusion of this 18 °C and are fed at least 5 d per week, with when the animals are maintained in the dark at incorporates a 2-week period of conditioning from Genikhovich and Technau ( light as important inducers of spawning. and introduced shifts in temperature and and Technau coordinated water changes. ( weekly feedings with mussel ovary ( brine shrimp larvae ( feeding of the anemones with freshly hatched weekly schedule. The protocol involved regular protocol for spawning ( Figure 3 918 c a protocol excavating soft sediment Trowel, (Wildlife spade or Supply, Eckman grab cat. no. 197-C15)for Hip waders (optional; Dick’s Goods, Sporting cat. no. 4039311) MgCl Adult anemones (polyps) l Fresh mussels (optional; e.g., Artemia salina seawaterArtificial (ASW; Instant Ocean) ) The protocol described in the text is modified ) Hand and Uhlinger ATER -Cysteine (optional; MP Biomedicals, cat. no. 0219464680)

| VOL.8 NO.5VOL.8 2 (Sigma-Aldrich, cat. no. M-8266) I

| ALS Protocols for spawning 8 eliminated the use of mussel consisting of several hundred individuals on a square a on individuals hundred several of consisting cysts (available commercially from multiple vendors) | 2013 Artemia Nematostella 4 Nematostella published the first | natureprotocols ), in addition to b ) Fritzenwanker 7 Mytilus edulis . This protocol Nematostella on a roughly Mytilus and attempting to establish a lab a establish to attempting and

) and

, available infishmarkets) . - Nematostella

Lighting a Spawning Feeding water Change Temperature Lighting c Spawning Feeding water Change Temperature Day Day or to col to or

9 8 7 6 5 4 3 2 1 Artemia 8 7 6 5 4 3 2 1 Artemia - - -

Change • • • • dishes. culture in larger tens or hundreds of genetically diverse males and females together single spot that reliably harbors the anemones, and you can culture analysis, developmental you can all collect of your from animals a establishing a laboratory culture to produce abundant embryos for in interested If,you merely are to contrast, maintain. in you wish to regeneration accumulate clonal living stocks for each genotype use then would you population, test a within eliminate to variability genetic lines clone single on experiment perform to wish you If locations. different from collected animals individual rate Furthermore, when the establishing culture you will have to sepa distances. short over substantially fluctuate can anemone the of distribution spatial the because body pool same the in spots ple multi from sediment of amount consistent a sample repeatedly the of abundance estimate To field. accurately the in variables these measuring for (room temperature) culturing theanemone’sculturing food, nauplii( shrimp brine (Fisher scientific, inaring-stand supported cat. no. S47788)ideal for (~250 ml; funnel Fisher Scientific,Separatory cat. no. 13-678-604) spills) of possibility hole-punch to allow airto circulateand the minimizingevaporation while lidsare plastic storage bowls ideal. with a with perforated lids canbe The with 1/3-strengthSmall culture glass dishesfilled food ASW(Pyrex cat. no. 13-711-7M) Transfer theanemones (Fisher Scientific, pipettes for retrieving containers) for thesediment collecting Wide, shallow, lids(e.g., containers storage plastic tight-fitting food with Artemia Not specified 16–26 1 × perwee ° Artemia C k Optional Conditioning Mytilu Nematostella 18 Dark s ° 75% Artemia C Day b 1 9 Artemia 2 1 in a small body water,of you must 1 0 18 2 1 1 × Dark perweek/timingnot ° 8 7 6 5 4 3 C 1 2 specified Optional 1 3 Artemia salina 1 4 Induction Light Artemia 24 70–100% 20–25 70–100% ° 5

C Light 18–20

) °

16 C

° C - - © 2013 Nature America, Inc. All rights reserved. transfer the animals toculture dishes containing 1/3-strength ASW. 4| so that they are exposed to rain and sun (and top them off with dH in which the culture, forming a lacy film over the sediment). If you wish to maintain these marsh microcosms in a semi-natural state, of the microbial community; this is evident in a color shift (e.g., often, a filamentous white fungus will proliferate in the ecosystem will undergo a visible phase shift, with a precipitous die-off of many invertebrates and a pronounced alteration  3| 2| (B) ( objective isnot tosampleconsistent areas of the pool. Use option Bifyouintend tosamplesuch areas. 1| A PROCE mixed water anairstone, overnight with which oxygenates thewater and in total. As themanufacturer’s per recommendations, circulate thenewly dH literper of seawaterArtificial wounds. Wash your hands thoroughly after completing the collection process. gloves when you collect the yousediments, if haveparticularly any open to contaminants, wear youwaders if decide to stand in the pools, and wear contaminants from nearby point sources pollution. of To reduce exposure potentially pathogenic bacteria, such as not will be inundated. lowduring tide, as the be pools will shallower and the regions between pools foot. In low-lying marsh habitats, it is preferable generally to collect samples fluctuates between 15 and 30 p.p.t. Such pools can be generally sampled on soft, flocculent sediments rich in organic material. The commonly salinity sites for collecting sites Collection REAGENT SETUP • • • • • • • ? Rotary shakerRotary (optional; Step see 15) cat. no. R80150) PetriPlastic dish, 150mm(or similarshallow vessel; Fisher scientific, (Fisher scientific,Scalpel cat. no. 089275A) razor blade (FisherSingle-edged scientific, cat. no. 12-640) tubing to stone anaeration Aquarium the airpump(for aerating Incubator at~25°C maintained Incubator (e.g., Fisher Scientific, cat. no. at~18°C 97-990E)maintained A

nimal collection TROU

) ) CR (i) (i) C C Extract the anemones thatprotrude from the sediment ( Remove the lids from the containers. Allowthe sediment tosettleand the watertoclarifyfor 1hor overnight. Seal the containers and transport them backtothe laboratory. At asuitableestuarine habitat,collectthe surface sediment (i.e., the top3–5cm)according tooption A,ifyour I ollection ollection of sediment at specified, predetermined sites of a pool ollection ollection of sediment throughout a pool circumscribed bythe bucket. effort toavoid disturbing the substrate), the topfewcentimeters of sediment canthen beexcavated from the area level willnot overtopthe rimof the bucket.) Aftergently removing most of the waterwithasiphon orscoop(inan using ahacksaw. Pushthis‘holey bucket’ afewcentimeters deep into the sediment of ashallowpool(where the water consistent area withalow-techmethod, remove the bottomof atallplastic bucket (such asa5-gallonpaint bucket) these devices are designed toexcavate greater depths thanare required tocollect the scientific collection of coastalinvertebrates.  sediment submerged. T Transfer surface sediment into shallowcontainers using atrowel orshovel, along withsufficient waterto keep the A dedicated sampling device, such asanEckman grab, canbeusedtosampleaconsistent area. However, ingeneral, D I B URE CAL

LES CR 2 I O (35–36p.p.t.) to produce full-strength 2–5litersASW of

H

T STEP Nematostella Within its range, pools reliable are particularly I OOT CAL N. vectensis

For full-strength ASW, Instant dissolve Ocean 35.9gof Do not keep the containers indoors for more than a day or two. The composition of the sediment ! I

N CAUT STEP ● G

T IMI I . These pools featuregenerally still waters and ON Obtainthe requisite permitfrom the relevant local and/or national authorities inyourlocalityfor will continue to thrive for weeks or months, keep the containers outdoors with the lids removed, Marsh water may contain levels high of N

G 2h Artemia Vibrio and species chemical cultures) air attached via

S upplementary Video1 (20–22 °C). may stored be indefinitely incapped containers at room temperature intodispensed culture vessels asrequired. Full-strength or 1/3-strength ASW spigot easily sothatitcanbe diluted anintegrated and carboy with a plastic prepare filtered 5–15liters full-strength of ASWattimeandstoreone itin 1/3-strength anemonesASW for (11–12p.p.t.). culturing It isconvenient to the pH. Dilutefull-strength dH two one part parts ASW with allows theCO The resultingThe solution stored canbe indefinitely at room temperature. MgCl After thistime, discard any remaining culture. hatching.eaten by 48hof theanemones. within used shouldbe shrimp Brine collect theconcentrated nauplii, avoiding unhatched cysts, which are not to becomeswimming) concentrated thefunnel. atthebottom of Thereafter, incolor orange (which are andactivelyhatched bright naupliilarvae stone. usinganaeration aeration After 24–36h, cease aeration, allow the full-strength funnel. ASW inaseparatory Subject thesolution to continuous Hatching shrimp brine to appear location prefer 2/3-strength ASW Nova Scotia, Canada, from andindividuals salinity this wassoundatahigher At itwascollected, thetime apopulation thatwe isolated from Peggy’s Cove, anemones. of anew strain working when should optimizewith thesalinity Thus, 1/3-strength ASW represents point, auseful starting but theresearcher populations of atthissalinity,reproduction fecundity peak provided of evidence notall 2 O O if the water level drops precipitously). 2 (7%, relaxant wt/vol)  2

CR of the water to equilibrate with that of theair, thewater tothatof equilibrate of with thus stabilizing Nematostella I T I CAL

Although a laboratory study of study of alaboratory Although Add ~0.5 g of Add ~0.5gof ) using awide-bore transfer pipetteand show optimalreproduction atthissalinity. natureprotocols

Dissolve 7 g of MgCl Dissolve 7gof Nematostella Artemia 1 8 . . To samplea cysts to ~100 ml of cysts to ~100mlof

| 2 VOL.8 NO.5VOL.8 in 100 ml of 1/3 ASW. in100mlof Nematostella protocol 2 O to produce |

2013

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© 2013 Nature America, Inc. All rights reserved. and females into separate bowls, sothat youcanregulate fertilization more precisely. The sexes of the anemones cannot be 14| However, the fertilization process willnot besynchronous for all eggs. 13| ? 12| 11| have an inhibitory effect on spawning  water and replacing itwith fresh 1/3-strength ASW. 10| 9| 8| animals from Monday through Friday, allowing them to go without food on Saturdays and Sundays. increased when animals are well fed  conditioning, for example, when feeding the animals orchanging the water. daily, from Monday through Friday. Pleasenote that the darkness canbeinterrupted briefly during this14-dperiod of 7| Induction of spawning ? pipette orbydecanting their medium and replacing itwithfresh 1/3-strength ASW). 1 hormore, and then perform awaterchange (either bytransferring the anemones toacleanculture bowlusing atransfer in situations inwhich they regularly experience physical contact witheachother ( do not activelycompeteoverspaceorshow aggression toward eachother. Therefore, they canbehoused athigh densities containing 20–50individual anemones ( 6| water changes becauseof evaporation. tolerate the increase insalinitythatoccursbetween substrate isrequired. The anemones cancomfortably does not need tobefiltered orcirculated. No sediment or room temperature under ordinary indoor lighting. The water The culture vesselscanbekept onalaboratory bench at growth thandid 10,20,66,100or125%(vol/vol)ASW area, 33%(vol/vol)ASWsupported higher population sample of 1/3-strength ASW. Pleasenote thatinasmall study ona 5| 1–2 h per week L extended in a feeding posture. physical contact with each other and most of them have their tentacles with 1/3-strength ASW ~3 cm deep. Notice that the anemones are in close are shown in a glass culture dish measuring 10 cm in diameter and filled Figure 4 920 distinguished onthe basisof gross morphology; they canbeidentified histologically by examining the developing germ cells protocol ong-term ong-term maintenance of adult

TROU TROU

CR CR

(Optional) To increase the synchrony of fertilization and earlyembryological development, youmay segregate males Ifmales and females are housed inthe same container, the eggswillbefertilizedwithno additional intervention. Move the anemones tothe benchtop and monitor them every15–20minfor spawning. Incubate the anemones for 12–15hat20–25°Cunder bright light. Inthe afternoon, thoroughly cleanthe container holding the anemones and change the waterbydecanting the old | VOL.8 NO.5VOL.8 In the morning, before youseektoinduce spawning, feedanemones withfreshly hatched Change the wateronce aweek. Maintain the anemones in1/3-strength ASWinthe dark at18°Cfor 14d. Feedthem withfreshly hatched Feed the anemones once perweekwith Maintain the animals inculture dishes containing I I T T

| I I B B Adult CAL CAL LES LES Nematostella

H H STEP STEP Nematostella OOT OOT | 2013 Water changes remove ammonia and introduce additional oxygen into the culture. Anoxia and ammonia Although I I N N G G | natureprotocols collectedinageographically limited in culture. Approximately 50 individuals ●

T IMI Nematostella N G 15 d per spawning cycle Nematostella 1 6 . . However, it is not necessary to feed the animals every day. We typically feed the 1 6 . Fig. can continue to spawn during extended periods without food, fecundity is greatly Artemia 4 ). Pleasenote that

●

T . Add 1–2mlof concentrated nauplii toeach~150-mlculture dish IMI

4 N . G

Nematostella isa‘gregarious’ anemone, sothatindividuals Fig. 4 ). Allowthe anemones tofeedfor Artemia . Artemia

© 2013 Nature America, Inc. All rights reserved. same anemone is shown at 1 ( Figure 5 19| Induction of regeneration are timed tospawnonarotating schedule. animals for subsequent spawning. Ifweeklyordaily spawning isdesired, multiple anemone cultures canbeestablished that 18| rinse with1/3-strength ASW. Replace the culture waterwithafresh 1/3-strength ASW. developing embryos. Cleanthe glassware byscrubbing and repeatedly rinsing withwater(no detergents). Perform the final 17| been pulverizedwithamortar and pestle. metamorphosed polypscanbefedwithfreshly hatched tentacles willhavedeveloped, and the juvenilepolypswillspontaneously settle tothe bottomof the culture plate. Newly uniform layerof cilia and aprominent apical tuft,which forms oppositetothe mouth ( larvae. Within2–3dof fertilization, the planula larvawillbeactivelyswimming aboutthe culture dishpropelled byafairly strength ASWatroom temperature toallowembryogenesis toproceed. It isnot necessary tofeedthe developing embryosor 16| l gelatinous mass surrounding the eggs. Thisgoal canbeachieved byincubating the eggsfor 10–15minin4%(wt/vol) 15| fertilized for a few hours after spawning.  and willbecompletewithin afewhours. eggs, which willserveasafertilization chamber. Withno further intervention, fertilization willcommence withinminutes pipette totransfer 10–20mlof waterfrom amale-only bowl(where spawning hasoccurred) into the bowlcontaining the containing 1/3-strength ASW(the volume of the eggmass plusaccompanying ASWwillbe~5ml).Use aseparate clean segregation and afteranemones havebeguntospawn,useacleanpipettetransfer the eggmasses into acleanbowl separate culture dishes once they havebeenobservedduring spawning ( in the mesenteries of reproductive adults head and the oral portion of the body column were resected using a scalpel. -cysteine prepared in1/3-strength ASWwithgentle agitation onarotary shaker d a

CR Introduce asingle adult anemone ina150-mmplastic Petri dishcontaining 1/3-strength ASW. Returnthe anemones toconditions of 18°Ctemperature and darkness and repeat Steps7and 8 tocondition the Thoroughly cleanthe containers housing the recently spawned anemones toremove the remaining gametes and/or Transfer the fertilizedeggs(orentire eggmasses) into individual wellsof 12-wellculture platesfilledwith2ml of 1/3- (Optional) Ifyouwishtomonitor ormanipulate zygotes immediately afterfertilization, itisconvenient toremove the I T

I |

CAL Nematostella

STEP b Perform this fertilization step relatively quickly as the eggs will only remain viable and capable of being regenerating missing head structures. ( a ), ), 2 ( e ● b

), ), 3 ( T IMI c ), ), 4 ( N G 10–14 d c d ) ) and 5 d ( 1 7 . However, itismore practical tosimplysegregate males and females into e ) ) after the a – e ) ) The Artemia , buttheir abilitytofeedisincreased ifthe ? environmental stress. during regeneration asatestof how they tolerate may be be fedduring the regeneration process. Individuals to for 5–7d( 23| bearing the mouth and tentacles. equal-length parts, one bearing the foot and the other slicing the anemone cleanlyintwoapproximately 22| the dish. razor blade tothe animal, pinning itagainst the bottomof mouth and the tipof the foot, firmlyapplyasingle-edged 21| 1/3-strength ASW. induced bytreating the animal with7mMMgCl tentacles. Alternatively, anextended, relaxed posture canbe 20|

TROU

regenerate missing structures. The animal need not Maintain the bisectedanimal in1/3-strength ASW Draw ascalpelblade along the edge of the razor blade, At apoint locatedapproximately midway betweenthe Allowthe animal tofullyextend itsbody columnand

B subjected todifferent environmental conditions Fig. 2 LES Fig. H OOT a and ANTICIPATED RESULTS). Aftersex 5 8 ), allowing the cutends of the animal . I N G natureprotocols Fig. 2 b ). Within5–7d, twotofour

| VOL.8 NO.5VOL.8 Artemia protocol 2 prepared in

| have 2013

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© 2013 Nature America, Inc. All rights reserved. from 0.01to1.00across sites a study of genetic structure intenEnglish lagoons showed, using random amplification of polymorphic DNA(RAPD),that61% of individuals shared asingle multilocus genotype, which ranged infrequency a Steps Steps 19–23, induction of regeneration: 10–14 d Steps 7–18, induction of spawning: 15 d Steps 5 and 6, long-term maintenance of adult Steps 1–4, animal collection: 2 h ● T Troubleshooting advice canbefound in ? 922 Be aware thatsome natural populations, such asthatof Crane BeachinMassachusetts a 23 12 6 4 S protocol

TROU

tep b T

le le IMI | VOL.8 NO.5VOL.8 1 1 N B collecting Failure to obtain anemones while P all all occurs generated or no regeneration at An inappropriate structure is several weeks temperature changes for regimen of feeding and after following the prescribed Failure to observe egg masses and often produce copious mucus with their tentacles withdrawn keep their bodies contracted, as evidenced by the fact that they The anemones are stressed, | LES G roblem

Troubleshooting table. H OOT | 2013 2 I 0 N . G | natureprotocols

T being laid), it may lack males and they will begin to degrade within hours of fertilized (they will fail to undergo cell divisions are being produced but the eggs are not being Your culture may lack females or, if egg masses A decline in water quality is a likely cause wound head or a second foot may form at the site of the If the animal is only partially bisected, a second in a two-headed individual that lacks a foot cut end of the animal, resulting temporarily to the mouth, a second ‘head’ may form at the regenerate. If an anemone is bisected very close substantially delayed or the animal may not even following wound closure, regeneration can be If mesenteries protrude from the cut-site provided is not rich enough a in the same marsh may be devoid of anemones anemones per square meter, whereas other pools Some pools may harbor hundreds or thousands of tially from site to site within a given estuary. The abundance of P able The food source the anemones are being ossible reason 1 1 Nematostella . 9 1 4 , havebeenshown toconsist primarily orentirely of single-sex clones derived byasexual reproduction Nematostella : : 1–2 h per week varies substan

1

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container with fresh 1/3-strength ASW Transfer the anemones to a new collections between May and October densities in the summer. Attempt your populations achieve the greatest knowledge of the site. advice from someone with firsthand have already been reported, and seek collect animals at an estuary where they single pool. If practical, attempt to than taking multiple samples from a Try sampling of multiple pools rather always always below the level of the pharynx along the animal’s primary body axis, Obtain a clean cut approximately midway presence of uneaten food tend to degrade quickly because of the anemones, as the water quality will hours of feeding mussels to the water should be changed within a few pieces to individual anemones ovary, and use forceps to feed small Try mincing some fresh mussel, especially developing germ cells of the mesenteries, which house the determined by histological examination reproductive individuals can be to the naked eye. The sex of the The sex of S olution Nematostella 1 7 Nematostella is not evident 4 6 . . The —indeed,

© 2013 Nature America, Inc. All rights reserved. 7. 6. 5. 4. 3. 2. 1. c at online available is information permissions and Reprints interests. CO optimizing the protocols. developed by J.R.F. All authors participated in writing the manuscript and AUT Marine Biology. D.J.S. was also supported by a Warren-McLeod Graduate Fellowship in Related Research from the National Institutes of Health (no. F31 GM095289-01). supported by a Predoctoral Fellowship Award to Promote Diversity in Health- Foundation grant no. MCB-0924749 (T.D. Gilmore and J.R.F.). D.J.S. was A is availableNote: information in the Supplementary complete withmouth, pharynxand tentacles, aswellthe abilitytocapture prey and feed. The foot-bearing fragment willbeginerupting tentacles within36h;by72–96h,afullyfunctioning head willform, about 50minlater, onaverage ~170minafterfertilization occurs about120minafterfertilization which isasign thatthe males havereleased their gametes. When reared atroom temperature, the firstcleavage of the eggs masses are negatively buoyant. The waterincultures containing male anemones thathavespawned willoften turnturbid, heat. Atypical female anemone canproduce several hundred eggs, which are contained withinajelly. The jelly-encased egg fully extended inafeeding posture. 250-ml culture bowl( few days before itwillreadily eatthe when firstcollected, butthey willgradually assume apinktint asthey feedon the animals upand down incleanASWusing awide-bore transfer pipette. The anemones tend tohaveaslight brown tint and debris may adhere tothe body of freshly collectedanemones. These contaminants canberemoved bygently pipetting to useadissecting microscope oramagnifying glassaided byobliquelighting inorder todetect small individuals. Sediment in afeeding posture ( protocol. Ifanemones are present, they willreadily protrude afewmillimeters abovethe substrate and extend their tentacles Hundreds of ANT om/reprints/index.htm cknowle

M When adult polypsare bisected, the head-bearing fragment should fullyregenerate itsmissing foot within24–36h. Once youcreate the proper conditions, spawning should occur1–6hafterthe anemones are removed from the light and With regular feedings and waterchanges asdescribed above, 50–100animals canbecomfortably accommodated ina H PET pdb.prot528 dejellyingzygotes.of anemone Estuaries vectensis Nematostella 114 anemone sea the in anomalies some (1992). 169–176 anemone sea the of growth 1935). vectensis (2012). 158–161 annual first the from report a Genikhovich,Technau,&Induction G.U. starletspawningthesea of in anemone,sea distributed widely unique, The Uhlinger,K. & C. Hand, and fission transverse reproductionby Asexual Uhlinger,K.R. & C. Hand, reproduction,and asexual and sexualculture, The Uhlinger,K. & C. Hand, T.A.Stephenson, J.A. Darling, organism: model a Establishing Tarrant,A.M. & J.F. Ryan, A.M., Reitzel, I OR C I I

, 9–18 (1995). 9–18 , N CONTR PATE G G FI dgm . Nematostellavectensis,vitro in 17 NANC BioEssays D IB ents 1 N. vectensis , 501–508 (1994). 501–508 , (2009). UT et al. et RESULTS I I

AL This This research was supported by National Science ONS The British Sea Anemones Sea British The Rising starlet: the starlet sea anemone,sea starlet the starlet: Rising l I

. 27 NTERESTS

Cold SpringHarb.ColdProtoc. The collection and regeneration protocols were Stephenson: A review, new facts, and questions. and facts, review,new A Stephenson: , 211–221 (2005). 211–221 , Fig. S upplementary Video1 individuals canbecollectedfrom lessthan1square meter of soft substrate using the present Nematostella vectensis Nematostella 4

Nematostella The authors declare no competing financial ). Healthy adults willbenearly fullyelongated for most of the time, and their tentacles willbe Nematostella vectensis Nematostella fertilization gametes,ofand Vol. II. (The Ray Society,Ray (The II. Vol. meeting. online version online of version the pape

Artemia h 8 ttp://dx.doi.or . However, when embryogenesis iscarried outat18°C,the firstcleavage occurs . Biol. Bull. Biol. BioEssays . ). The animals canbeidentified withthe naked eye; however, itcanbe helpful http://www.nature. . Nematostella Invert. Biol. Invert. g/10.1101/

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theUnited Kingdom based onRAPD analysis. lagoonalsea anemone, fission. asexual of forms two and vectensis Nematostella anemone sea starlet the in trajectoriesdevelopmental alternate activities. vectensis Nematostellavectensis 1997). Davis, California, of (University 1935 Stephenson, vectensis Nematostella Anemone, Sea http:/ evidence for local adaptation to high temperatures. high to adaptation local for evidence anemone sea estuarine the by temperature England. New in Stephenson invertebrate: estuarineintroduced widely a of 1983). 43–46(International UnionConservationfor NatureofNaturaland Resources, Aquatic Conserv.: Mar. Freshw. Ecosyst. Freshw.Mar.Conserv.: Aquatic vectensis Naturalist anemone the (2008). 1197–1213 anemonestarlet the ofstructuregenetic Evol. Genes Dev. in fission asexual regenerationand between (2002). 99–103 cnidarian basal introductions and implications for conservation policy.conservationintroductionsimplicationsfor and Pearson,C.V., Rogers, A.D. Sheader,& M.The genetic structure ofthe rare of Comparison Finnerty,J.R. & C. P.,Krone, Burton, A.R., Reitzel, J.C. Sullivan, Bleakney,Frank,P.& Histologysexual J.S.andreproduction anemonetheof Uhlinger,K.R. Reitzel, A. Reitzel, structure population Regional Finnerty,J.R. & A.M. Reitzel, J.A., Darling, Williams,R.B. anemone,The G.A. Rowe, & A.M. Suwailem, Sheader,M., of anomalies and reproduction,diet, Asexual Bleakney,J.S. P.G.& Frank, Reitzel, A., Darling, J., Sullivan, J. & Finnerty, J. Global population Global Finnerty,J. & J. Sullivan, J., Darling, A., Reitzel, expressiongene novel and Conserved Finnerty,J.R. P.M.& Burton, the in gametogenesis ofInduction U. Technau, & Fritzenwanker,J.H. /dx.doi.org/10.3354/ are widely dispersed in nature and encode proteins with distinct with proteins encode and nature in dispersed widely are , in Britain: considerations for conservation management.conservation considerationsfor Britain: in ,

92 PloS ONE PloS et al. et , 259–263 (1978). 259–263 , Nematostella vectensis Nematostella et al. et Nematostellavectensis Sexual Reproduction and Early Development in the Estuarine the in Development Early and Reproduction Sexual Artemia

Nematostella vectensis Nematostella Physiological and developmental responses to responsesdevelopmental and Physiological 219 Two alleles of NF- of alleles Two

4 , 79–87 (2009). 79–87 , Stephenson 1935. , e7311 (2009). e7311 , Nematostellavectensis (Stephenson): embryogenesis, regeneration,embryogenesis,(Stephenson): . It may take afield-caught animal a natureprotocols meps1028 Mol. Ecol. Mol. Invert. Biol. Invert. . in . in Nova Scotia. Nova in 2 ().

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The IUCN InvertebrateIUCNThe BookDataRed B in the sea anemone sea the in B 13 , 13–25 (1997). 13–25 , J. Nat. Hist.Nat. J. Nematostella vectensis Nematostella Stephenson (Cnidaria; Anthozoa) in , 2969–2981 (2004). 2969–2981 , Mol.Ecol. Nematostella vectensis Nematostella Nematostella vectensis Nematostella

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