© 2016 Nature America, Inc. All rights reserved. acteristics that facilitate its use in laboratory-based research. research. laboratory-based in furzeri N. use its facilitate that acteristics reports by corroborated laboratories other from observation an months, 17 reached spans life shorter longest-lived even have lines inbred Established months laboratory-bred <6 is wild-derived of span life fly’ hatching fruit ‘vertebrate of role the laboratory assumed the in cultured be can that vertebrates furzeri N. Advantages of the of periphery southern furzeri N. species ~70 comprising genus African the of members by exhibited is aging ver tebrate typical with associated deteriorations functional undergo same the and conditions laboratory under lives short similarly live intrinsic—they is span life short This habitat. their of cation span, only a typically few months, is limited by the seasonal desic life posthatching their where America, South and in Africa pools in a under well freshwater year. live in temporary killifish Annual span life entire their complete killifish annual many contrast, By agendas. research many for practical be to long too live simply models. Vertebrates animal non-vertebrate span— with compared short life trait—the history fall vertebrates, other as all as well species, model life fish traditional particular one of terms in become fundamental models in particular disciplines promelas research ( medaka and models such as zebrafish ( fishes have become proven model taxa. Traditional fish laboratory a with and physiology output, several reproductive and high culture of ease relative and plan body vertebrate a combine Fishes I nonaquatic embryo development, with consequences for their laboratory culture. requirements of everyday care of juvenile and adult fish, breeding and treatment of most common diseases. for the maintenance and breeding of the species under laboratory conditions. We provide details for egg incubation, hatching, in maximum (90%) life span of 9 months. T Published online 7 July 2016; Institute of Vertebrate Biology, Academy Sciences the of of Czech Republic, Brno, Czech Republic. Correspondence should be addressed to M.P. ( Matej Polacˇik, Radim Blažek & ReichardMartin killifish Laboratory breeding of the short-lived annual 1396 very is it pools temporary space-limited in origin its of because NTRO urquoise killifish, protocol The short life span of of span life short The Among fish, medaka, guppies and zebrafish all live ~4–6 years N. N. furzeri

| VOL.11 NO.8VOL.11 D UCT 1, ad h sikeak ( stickleback the and ) is the species possessing the shortest life span among among span life shortest the possessing species the is Jubb, originating from a relatively arid region in the the in region arid relatively a from originating Jubb, is a small fish (maximum length 7 cm; cm; 7 length (maximum fish small a is 2 Ohr sc a te aha mno ( minnow fathead the as such Others . becoming a widely used laboratory model species in aging research and other disciplines. Here, we describe a protocol I 7 ON 11,1 . Among the annual killifish, the shortest life span span life shortest the killifish, annual the . Among . furzeri N. N. furzeri rza latipes Oryzias N. N. furzeri 2 , only twice that of of that twice only , | 2016 Nothobranchius Nothobranchius furzeri Nothobranchius furzeri 8 1 doi:10.1038/np | , with the shortest life span reported for for reported span life shortest the , with

4 N. furzeri N. as a lab model Danio rerio individual in our breeding facility facility breeding our in individual natureprotocols . substantially differ substantially from those of other fish model taxa; Nothobranchius ) are widely used in biological biological in used widely are ) is combined with other char other with combined is atrses aculeatus Gasterosteus ), guppies ( rot.2016.08 Drosophila melanogaster Drosophila T his his short life span, which is unique among vertebrates, evolved naturally and has resulted , , have an short intrinsically life span, with a median life span of <6 months and a range. 1 0 te ein post median the ; 0 Poecilia reticulata Nothobranchius 9 3, Fig. Fig. , and it has has it and , Pimephales Pimephales 4 . However, . furzeri N. 9,1 1 ) have have ) 1 ), and and ), . The The . 2,5, 1 3 6 ) - - - - . , .

Dorsal Dorsal and anal fin are also relatively larger than in a female. temporary colors preceding the appearance of red, yellow and blue colors. it tolerates water temperatures from 15 to 35 °C, including rapid rapid to °C, 15 35 including from temperatures water tolerates it savanna), Mozambican (southern distribution natural tropical sub its of result a As space. aquarium of terms in undemanding opalescent opalescent hue on flanks and black pigmentation in the dorsal fin in ( Figure 1 weeks <3 in reached is maturity hatching, Upon are wetted. eggs the when achieved be can cohorts experimental of ing hatch and synchronized at stage) this shipped can be (and easily diapauses is embryonic 3 the result three of facultative years; this variability and d 17 between varies development 30–50 day. each Embryonic eggs produces female single a rations, food optimum Under after paired, (often being seconds) and is the prolific. very species reproduce on a daily basis. A pair begins spawning within minutes and bold naturally are fish the because feasible are behavior, ing fluctuations genesis ogies, including abundant occurrence of tumors strain viable perfectly including existence of and an annotated brain atlas strains, (homozygous) inbred inbred fully various one of production the include a c a ); ); red form, male ( Methodological innovations in laboratory research on 21–2

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( . Behavioral observations, including those of mat of those including observations, .Behavioral Fig. N. N. furzeri b ); ); adult female ( 2 Nothobranchius ). Eggs are incubated in a damp environment E live brief lives and in nature undergo mphasis mphasis is given to the fact that the c

furzeri d b ); ); and maturing male ( 2 0 ; description of histopathol . . ( a – d ) ) Yellow form, male [email protected] 2 7 4 ; successful trans ; age-dependent age-dependent ; d ). ). Note N. furzeri ). 1 8 . d , 1 9 - - - - - ;

© 2016 Nature America, Inc. All rights reserved. influence of ambient temperature, food rations and housing housing and rations food temperature, ambient of influence laboratories among trait parameters history life and span life of estimates inconsistent include of limitations Current of Limitations potential with medicine human in application mechanisms regulatory underlying opportunity the identify offers to also it work, laboratory for history life lines. their of aspect undesirable non-inbred an as viewed be in might this Although especially conditions, laboratory trolled con despite variability phenotypic generate that norms reaction broad relatively their by extends manifested is and breeding feature captive into This change. environmental an to ‘sensitivity’ environment their to responses work— laboratory events chance exploit to abil ity the is principle prevailing the which in environment able traits life-history environment its to adaptations as naturally evolved have animal experimental able make that features technology CRISPR/Cas9 genome-editing using toolkit and genomic a of tion genome bled microRNA expression profiles DII, diapause II ; DIII, diapause III. with ambient temperature. DI, diapause I; developmental processes varies greatly at 25 °C). Note that the exact timing of of Figure 2 a d N. furzeri Another advantage is that the specific specific the that is advantage Another

| Developmental trajectories in aquatic medium (egg incubation 26,2 N. furzeri 7 ; and successful applica successful and ; 3 4 Nothobranchius N. furzeri N. Tmoay ol rpeet n unpredict an represent pools Temporary . 15,17,18,29–3 . furzeri N. as a lab model such a valu a such 2 3 b 5 3 4 ; an assem , with natural trade-offs among among trade-offs natural with , . This also brings a caveat for for caveat a brings also This . 3

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Variable duration,typically several brands of widely available available widely of brands several using food juvenile for achieved be can standardization of level satisfactory relatively a Although laboratories. individual within zation of food quality among laboratories and across experiments second A limitation is the lack of diet artificial protocol. that would enable standardi standardized and accepted commonly a of of span life and furzeri N. reproduction growth, the on housing) vidual indi versus water,social of volume per density (fish conditions enabled by definitive hosts such as birds. In captivity, captivity, In birds. as such hosts definitive is by enabled pools temporary between transmission their and season dry the during parasites the of survival cycles: life complex with sites wild, the In of Diseases available. currently bloodworm live and ( diet—frozen adult the of content energy frozen food from sources with no guarantee of a pathogen-free pathogen-free a of guarantee no with sources from food frozen infected with a pathogen is greatly increased by the use of live and commonly suffers from ( several diseases several daysorweeks Chironomus Fertilized eggs 4–6 9–11 d , these inconsistent estimates are partially due to a lack lack a to due partially are estimates inconsistent , these d DI N. furzeri Nothobranchius larvae)—are locally variable. No artificial diet is is diet artificial No variable. locally larvae)—are DI Direct (escape)development17–21 I Variable duraon,typically several daysorweek development typically severalmonths Post-DI natureprotocols Variable duration ( as protruding eyes—exophthalmos) and unspecified bacterial infection (here manifested the body ( velvet ( N. furzeri by by common Figure 3 dinoflagellate, a by caused is which (velvet), oodinosis is disease common most the By far product. nostics and treatment of oodinosis, see see PROCEDURE). the of 25 Step oodinosis, of treatment and nostics diag (for conditions deteriorated under triggered outbreaks with time, of periods for shows long no symptoms populations, infection is latent in captive oodinosis the that possible also is It food. live or contact mutual through infected Fig. 3 dots on the fish’s body and fins miniature of consisting coating yellowish pillulare P. d ) ) N. N. furzeri development Post-DI 5–7 a spp. are typically infected by para by infected typically are spp. P. pillulare) with with

male ( | d External appearance of fish infected b s ); ); ( N. N. furzeri DI male showing symptoms of dropsy. infestation causes a sort of of sort a causes infestation I Artemia Fig. Fig. 3 c ) ) a development Variable duration,typically ); ); N. N. furzeri male Post-DI —note the yellowish spots on

N. N. furzeri Piscinoodinium pillulare Piscinoodinium 13–16 | d Fig. several months VOL.11 NO.8VOL.11 pathogens. ( b development ). The fish can become become ).can fish The Post-DI I eggs, the quality and and quality the eggs, d 3 ). The risk of being male infected with I protocol 8–10 Nothobranchius infected with 8–10 a (ready tohatch | d 3 – 2016 7 d d (compare ) ) Healthy N. furzeri N. DIII

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© 2016 Nature America, Inc. All rights reserved. T stocks. entire destroy to potential the has it occurs, it When growth ( cells host the inside ‘xenomas’ water insufficient change). (e.g., poor is care cause fish when and only ubiquitous problem a are agents the of Most the of 26 PROCEDURE). Step see diagnostics, (for affected are stock the in als viruses and bacteria including agents, several by shared are dropsy of toms ( dropsy as such diseases Particular care. optimal otherwise under are fish when even PROCEDURE), the of 28 of Step see treatment infections, and unspecified diagnostics the some (for with changes together appetite, morphological and activity a in as decrease manifest These general origin. bacterial supposedly of infections courtesy of Alexander Dorn. of Figure 4 1398 able able Median laboratory life span Maximum laboratory life span Intraspecific aggression Specific spawning substrate Spawning periodicity Age at maturity Food quantity requirements Adult artificial diet Raising juveniles on an artificial diet Onset of feeding after hatch Hatching success Embryonic development A protocol Glugea . furzeri N. spect of husbandry

| VOL.11 NO.8VOL.11 1 1

3 sp. are visible as white cysts marked by a red ellipse. Photograph | 8 | Male (for diagnostics, see Step 30 of the PROCEDURE). PROCEDURE). the of 30 Step see diagnostics, (for Glugea

Comparison Comparison of key aspects of husbandry between 3 fed unsound food may suffer from unspecified unspecified from suffer may food unsound fed 7 N. furzeri , and typically only a small number of individu of number small a only typically and , Fig. 3 Fig. sp. is a unicellular microsporidian that forms forms that microsporidian unicellular a is sp. | 2016 infected with d ) and and ) | natureprotocols Glugea Glugea Fig. Fig. are untreatable. The symp The untreatable. are 4 sp. Xenoparasitic complexes ), which causes parasitic parasitic causes which ), 0.5 0.5 year 1.5 years risk of injury Male aggression—serious Not required (30–50 eggs daily) Multiple events each day 3–4 weeks High Currently unavailable Currently unavailable Immediate Variable Erratic N. furzeri 11,1 N. N. furzeri 2 - -

, , zebrafish 3.5 3.5 years >5 years Negligible risk of injury Not required couple of days (100 eggs per spawning) Potentially daily, typically once in a 12–16 weeks Normal Available Available but suboptimal Delayed Stable Predictable and among studies of aging were also performed among controllinglife span pattern of microRNA expression topathology associated with aging including studies of early embryogenesis experimental animals in laboratory studies in the 1970s and 1980s, Some for applications Experimental are in progress. Notably, the short generation time makes makes time generation short the furzeri N. Notably, progress. in are sympatric species growth compensatory evolution and ecology into strategies life-history and diapause ment in bet-hedging developmental of function activity telomerase high despite shorteningtelomere aging-related of demonstration tion of histopathologies including abundant occurrence of tumors cations that used laboratory breeding of lower ambient temperature Furtherstudiesshowed that extend life span and retard the expression of age-related markers model to demonstrate that resveratrol (a natural antioxidant) may gestion to use natural life span of after the publication of a study that demonstratedA renewed the interestextremely leading shortto intense scientific attention started only Laboratory breeding of of breeding Laboratory Nothobranchius D. D. rerio 5 4 an apposite model for experimental evolution studies. evolution experimental for model apposite an , age-dependent decline in adult neurogenesis N. furzeri D. rerio N. furzeri and medaka 6 0 N. furzeri 5 have been completed, and several other studies 1 3 5 64,6 7 , aging-related impairment of mitochondrial of aging-relatedimpairment, 6 and related species , which led to within-population alternative alternative , led to which within-population species, especially 3 andregenerative capacity in aging research 4 5 . 5 9 N. furzeri N. and on reproductive isolation among among isolation reproductive on and 5 N. furzeri N. in 2003 (ref. 1 1 O. latipes. 9 and dietary restriction N. furzeriN. ; studies on resource allocation and and allocation resource on ; studies 2 N. furzeri 5 , mapping of quantitative trait loci 45,4

has great potential in research research in potential great has N. furzeri N. 6 was further used in studies studies in used further was , toxicology aging is also deceleratedalsois agingby 4 12,5 39–4 9 N. furzeri N. guentheri 9 . It was the first vertebrate ). This prompted the sug 2 2 years 4.5 years Low risk of injury Required Daily (1–14 eggs) 6–12 weeks Normal Available Currently unavailable Delayed Stable Predictable 8 1 . N. furzeri embryonic develop embryonic , diapause O. latipes 14,5 4 7 include descrip and behavior 7 5 .Comparative 2 , were used as . Other appli lab strains 5 16,42–4 66–6 5 , temporal 9 4 , his 5 4 1 0 8 7 - - - - - 1 , . .

© 2016 Nature America, Inc. All rights reserved. sufficient egg reserve and rapid produc rapid and reserve egg sufficient a with overcome be can problems initial food high-quality of amounts are large fed fish parental when quantities large in obtained easily be the can Eggs fish. raising and hatching development, embryo with problems potential any gate to miti eggs of number a large producing keeping When such and breed, to easier as are that species with experience initial gaining ommend using research term rate. hatching actual in variability substantial in results ( conditions incubation the often asynchronous ( products. Their embryonic development is water exchange to remove metabolic waste rations, more nutritious food and frequent food greater need they fast; very live and that of zebrafish or guppies. which the culture of in respects important the of appreciation of lack a from stems often Wethis believe initial for reasons that have not many been determined. these, attempts to establish a breeding colony fail Of laboratories. receive for often requests we wild, the from lations small of other number a from imported difference ( their fishes laboratory accept and recognize to diseases. common most its curing and eggs its incubating breeding and provide raising we hatching, for section, details PROCEDURE the In laboratories. across declines or aging-associated survival of such cedures as estimates pro experimental particular standardize to intended not is and using in work order to experimental of promote greater reproducibility laboratories across applicable protocol current to the make tried CO the (e.g., husbandry and laborious unnecessarily in inapplicable large-scale laboratory or container) new a to fish hatched freshly the of transfer (e.g., unnecessary hatching), promote to water of body deep a in eggs have found some of the advice therein ineffective (e.g., sinking the http://www.nothobra at (available T. by Genade manual the than compact more be to intended is and practices laboratory common emphasizes tocol effective be to proven consistently have presented herein methods and procedures All purposes. scientific for 10 in years experience breeding of first-hand This the procedureof Overview tion of a new generation of fish. of generation new a of tion For those seriously interested in long- in interested seriously those For of culturing successful for factor key The breed and maintain to how describes protocol This ohbacis korthausae Nothobranchius ohbacis guentheri Nothobranchius . furzeri N. N. furzeri N. . furzeri N. N. furzeri N. as a model. a as laboratory husbandry protocol is based on on based is protocol husbandry laboratory Table N. furzeri Fig. nchius.info/pdfs/lab . furzeri N. eggs from other other from eggs 2 N. furzeri N. , we recommend recommend we , exhalation hatching method). We method). have hatching exhalation 2 1 ), influenced by N. furzeri ). As we have have we As ). Fig. Fig. differs from 18,5 Meinken Meinken 5 w rec we , Pfeffer. Pfeffer. ), which which ), 9 popu . Most Most . grow - - - -

_protocols_1.pd N. furzeri N. Nothobranchius of Figure 5 Nothobranchius diapause skippers 18,34,5 N. N. furzeri. (Step 16A(vi)) synchronized development Incubation at hatch eggs 27–28 °C Ready to 15–20 din Fast,

Group spawning(2weeks)(Step15A) 9 | , as well as as well as , . The pro The . Schematic overview of the sequence and duration of different steps in the husbandry regime N. furzeri N. Eggs inpeat(filterpaper1–3d) BBS, baby brine shrimp. f ). We spp. (Step 16A) is is (Step 16A(vi)) 2–16 months Incubation at development - - hatch eggs 22–25 °C

Ready to Erratic The protocol can be modified to effectively use locally locally use the is outcome effectively of expected to the maintenance that modified Given resources. be available can protocol The design Experimental and and Step the 16 PROCEDURE (see of performance and posthatching success for hatching consequences important with development, of pace different a to embryos predisposes each and cumstances rate mortality embryo damp on effect their of in vary top methods The peat. on damp of inside and medium, peat aqueous incubation—in embryo incubation. Embryo of analysis traits. for functional consequences potential with offspring, their of been expression state phenotypic the into be translated can fish nutritional parental of and have social the that assume epigenetic) should researcher or fishes annual (maternal in departure trans- reported Notably, effects substantial stated. clearly any be generational and should protocol taken, this from be However, implement. should to modi caution straightforward such be assays, should experimental fication direct than rather animals diapause) tend to live faster and shorter lives than embryos that that embryos than lives shorter and faster live to tend diapause) Fig. Fig. 17–20 °C(1month) Wean onchoppedbloodworms,10 Wean onfull-sizebloodworms,15 (Step 16A(vi)) (synchronized development) incubation at development (4–6 weeks) 5 Incubation at hatch eggs 24–26 °C for details). Embryos that develop directly (without (without directly develop that Embryos details). for Resume Ready to Adults (3–4weeks)(Step12) Eggs readytohatch(Step2) Halted Juveniles (1–12h)(Step6) Shipped eggs(Step1) (10–15 d)(Step10) Advanced juveniles with BBS(12h) Start feeding . furzeri N. Check Hatch natureprotocols We provide three basic alternatives for for alternatives basic three provide We (Step 16A(vi)) development Temperature- hatch eggs See group controlled spawning (Step 16B) In thepeat Ready to 6 Pair spawning(2dconditioning)(Step15B) 1 1 8 , and therefore as a precaution the the precaution a as therefore and , and production of experimental experimental of production and ec i ueu udr eti cir certain under useful is each ; Eggs inpeat,onpeatorwater

d d within 3–4weeks hatch-reluctant (Step 16C(iv)) eggs andbelly At 28°Cmost eggs develop faster; higher (Step 16B–D) On thepeat proportion of (Step 16C) hatch eggs Relatively Ready to

sliders | VOL.11 NO.8VOL.11 protocol peat atDIIorDIII reluctant eggs Transfer tothe (Step 16D(xi)) stage (9–21d) faster, hatch- In thewater | (Step 16D) 2016 Relatively hatch eggs Ready to |

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© 2016 Nature America, Inc. All rights reserved. • • • • • • • • • • • • • • • • • • • • • • • • REAGENTS M we as far As possibilities. only the as food live and frozen leaves for diet artificial an furzeri N. of dity Feeding. versa). vice (and temperature ambient higher at accumulation) higher product waste with faster (and temperature, requirements food ambient with scales rate metabolic by tolerated produc from tion. 24 However,to ranging 32 egg °C are readily temperatures and rate growth fish enhance to °C 28 at perature tem water maintaining We recommend PROCEDURE). the of 25 Step (e.g., infections particular to prone more subsequently furzeri N. by tolerated is conductivity) lower (with water Soft pH. alkaline with salts) and magnesium calcium of concentration water (high of parameters these fluctuations tem of frequency chemistry, the as water well as depth including and perature, quality, in widely vary that quality. Water papers. research of Methods the in stated be method mental diapause via developed are 1400 protocol has been certified by the Ministry of Agriculture of the Czech of of Agriculture Republic certified has been by the Ministry relevant institutional and nationalregulations. facility Ourhusbandry by post annual are killifish suitable for drought-resistant shippingThe of eggs stock,laboratory breeder a specialized from or imported thewild. N. furzeri (Tetra, testChlorine strips cat. no. 19542-00) Gentamicin sulfate (in10mg/ thiosulfateSodium (in5g/l solution; P-Lab, cat. no. T05302) Wear protective gloves handling. during acids. incontactwhen with Avoid eyes, contact with skinandclothing. ! 5% sodiumhypochlorite s Methylene blue (0.002g/lsolution; P-Lab, cat. no. R15141) seaalmond Dry NaCl solutions (4, 8–10, 100and360g/lsolutions; P-Lab, cat. no. R39573) test; (chlorine EasyStrips Tetra, cat. no. 19544-00) OxyTabs cat. (JBL, no. 20080) (e.g., fish orfood granulated Exot Hobby, SAK55andEnergy) item) food (supplementary food Dry Live choice local zooplankton of Live mosquito larvae Live worms ( glass shrimp Live fairy Live bloodworms Frozen zooplankton (Hikari; cat. no. 30320) Frozen ( shrimp fairy Frozen Tubifex worms ( bloodworms, (cat. no. 30240) Frozen regular-size bloodworms ( Premium Artemia (Sanders Shrimp) Brine Artemia salina (e.g., beads; P-Lab, 0.25–0.5-mmglass cat. no. R155361) adiameter for (substrate upto pairspawning)Glass beads with 0.5mm cat. no. EAN8594005007192; Reagent see information) for Setup further Peat: pH6.5for growing orchids mixture apeat of (Agro CS, Chlorine-free fresh water ATER

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| VOL.11 NO.8VOL.11 I N. furzeri I ! ON ALS

Feeding has an important effect on the growth and fecun killifish. obtained be The fishcan from anestablished CAUT welfare and standardization. Despite ongoing efforts, efforts, ongoing Despite standardization. and welfare , but it has a lower buffering capacity and the fish are are fish the and capacity buffering lower a has it but , Bleach isastrong corrosive, anditreleases toxic chlorine eggs (baby brine shrimp (BBS))juvenile (baby shrimp brine eggs food. Sanders I . furzeri N. ON Terminalia cattapa In the wild, wild, the In Chaoborus . Any involving experiments live fish must with comply 1 Ad libitum | 5 2016 Artemia . In captivity, we recommend the use of hard hard of use the recommend we captivity, In . Tubifex N. furzeri N. olution (household bleach; Savo) . It is important to keep in mind that that mind in keep to important is It . |

spp.) natureprotocols spp., cat. no. 32464or l solution; P-Lab, cat. no. D.02-012E) spp.; cat. no. 32920) 3 feeding is feeding the approach preferred for 4 . We urge that the embryo develop embryo .the Wethat urge N. furzeri N. Chironomus leaves (e.g., ITPhilippines) is not currently available, which which available, currently not is . Animal-protein-based pelleted dried inhabit bodies of water water of bodies inhabit spp.), not ‘jumbo’ Daphnia

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mended. The extra handling required diminishes most benefits benefits most diminishes required handling extra The mended. may fish for eggs be female fatal lay to failure the and capacity, reproductive retain to eggs ovulated spawn to need fish spawning—female for fish of ment move frequent requires housing individual that emphasized be must It absent. not although lower, are rates growth individual among differences individually, use. are housed for When fish wider it recommend cannot we and malfunctions, frequent suffers from but inexpensive relatively is system This (AquaMedic). system zebrafish FishBox the with professional experience We have used. be can of sufficient. racks is range a system purposes, recirculation experimental a For without aquaria of set a or via chemical communication. However, for regular husbandry, encounters direct through either individuals, subordinate of rate growth the decrease interactions Social rates. growth individual conditions. Housing breeding. and for necessary not is it recommended, is assays experimental major in use its While high. relatively is cost the and country by varies availability its although environment, and infection-free controlled in a cultured supposedly artificially are aware, only a product Bio-Pure single (Hikari is bloodworms) 450–550 450–550 fresh Chlorine-free water REAGENT SETUP • • • • • • • • • • • • • • • • • • • • • • EQUIPMENT housing. individual of Timer AquaMedic, cat. no. 403.10) Recirculating fish(e.g., system FishBox; for housingof individual Scientific,Thermo cat. no. 3915FL) incubator (e.g.,Cooled laboratory Peltier cooled incubator; White cellulose filter (40×40cm; paper Merci, cat. no. 480622080040) (0.5liter; washbottles P-Lab,Laboratory cat. no. K001638.N; Glass ‘pipe’ for manipulating fishAlpha Laboratories, cat. no.(4 LW cm 4060; in diameter; Sklorex, cat. Fine Tip Pasteurno. to tip open to 3–4mmdiameter; Pipettes (cut off 531–2; 1mmmesh (e.g.,Sieve with atea strainer; and round (diameter 10–20cm)Petri dishes (10×10cm,Compartmented 5×cells; P-Lab, cat. no. S000103; Standard andteaspoon diningspoon Ento Sphinx, cat. no. 21.33) Fine entomological tweezers (soft grade, 0.2-mmsteel thickness; Parafilm (P-Lab, cat. no. P701605) Plastic zipper bags (5 × 8 cm for shipping eggs, 15 × 20 cm for egg incubation) Glass jars for group spawning (volume or boxScalpel knife 0.6 l, minimum 8-cm-wide opening; tubs (containersPlastic lids, with volume: 2l; cat. no. 6042100) submersibleThermostat-equipped heaters aquarium (25–300 W; JBL, Hand (0.15–3mmmesh) nets 12-mm-Diameter tubingcat. aquarium (JBL, no. 6108200) 4-mm-Diameter cat. (JBL, airtube no. 6108500) (l ×h)(Xinyou; Free-standing air-driven sponge filters, e.g., model XY 2837, 16×15cm Glass aquaria, 6–80l (M.P., CZ01283; R.B., CZ01265; andM.R., CZ01285). theCzechAgriculture Republic of of animals issued by theMinistry 1/2016). authorsAll holdcertificateswith forexperimental working by Academy theCzech Sciences Republic of of (125/2010, 138/2010and (43245/2008-10001 and43245/2008-17210). procedures The were approved µ S/m 2 , 7.0–7.5 pH) water for all in applications the protocol. Fig. 6 ) Fish housing conditions and density affect affect density and conditions housing Fish

Use hard general (15–18 hardness,degrees of 3 4 . Spawning twice per .week is twice recom Spawning Fig. 6 Fig. ) Fig. 6 N. furzeri N. ) 6 ) maintenance maintenance

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6 6 ) ) - - © 2016 Nature America, Inc. All rights reserved. than than 5 min, to avoid their desiccation.  embryo ( search for the eggs. Eggs willbetranslucent withno apparent structures inside ( 25–40 W)toinspect the eggs, which are >1mmdiameter and canbeseenwith the naked eye. Gently stirthe peatto of peatinaplastic bag)and pourthe peatinto aPetri dish.Use adirected light source (e.g., standard tablelamp, 1| Hatching (shipped)eggs PROCE upwardwith pointing outflow ( 2 l per fish. Equip the tank an with air-driven, free-standing, removable filter 3–4 d, see Step 8 the of PROCEDURE) in a tank a with minimum volume of Aquarium setup for juvenile fish EQUIPMENT SETUP the fishhatching isplanned. fishhatching. incubating of time Start available nauplii. N. furzeri thesaltsolution do aproblem.remnants notpose of Freshly hatched Use aPasteur pipette (eye dropper) to BBSto transfer thefishaquarium; BBS into (diameter aseparate(air)tube tub usingaplastic 4–5mm). float to the surface (~4min). thebottle. will shells of Empty Siphon out Remove andallow tube theaeration hatched BBSto atthebottom aggregate incubation.23–28 °C. of BBSbetween 24and36hafter Collect thestart (anairstone thebottle to helps holditthere).of Incubate themixture at needed.conditions Introduce if airflow strongbottom atthe with aeration (Sanders Shrimp), Brine inourlab. used Adjust to local quantity theegg conditions; hatching success Sanders Premium is90%with Artemia commercially of available thebrand with varies three (one times teaspoonful is2.9–3.2g). Note thathatching success naupliisufficient atleast1,000freshlyto feed amount of hatched (assume three events) feeding inabottle. to Use produce eggs an 3gof fish to feed depending on thenumber BBSeggs of salt andanamountof A. salina beforeits acidity useandto make by air. itsinkrapidly removing trapped repeatedly necessary). thepeat to (2–3times) Boilandrinse lower if particles wood or suchgravel aspieces (useasieveparticles of to remove hard ncubation. Ensure contains thatthesubstrate or fertilizers noadded hard plants;specific some have pHandare higher suitable for spawning and suitable. There are many gardening peat-based intended substrates for any suitableavailableof brand worldwide. Peat pH(>5)ismore higher with Peat Gentamicin sulfate thiosulfateSodium solution Methylene blue solution NaCl solutions Wear protective gloves handling. during acids. incontactwhen with Avoid eyes, contact with skinandclothing. ! hold bleach diluted shouldbe to 1ml/0.2lwater and10ml/100lwater). solution hypochlorite Sodium lower byagainst potential acidification capacity buffering peat. (see certain infections Step 25 the of PROCEDURE) and soft water has a Soft water (50–300 for catching and transferring juvenile fish, and (9) laboratory wash bottle. (tea strainer) with 1 mm mesh, (7) disposable Pasteur pipette, (8) glass pipe peat, (5) plastic incubation Petri dish with 25 compartments, (6) sieve glass beads, (3) spawning tub with mesh, (4) spawning jar filled with (1) Free-standing air-driven sponge filter, (2) spawning tub with fine Figure 6 the peatissubstantial (>150–200ml), check onlyarepresentative sampleof the batch(e.g., 20%of the peatvolume), as 2|

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This is a highly locally diversified locally isahighly This arecommodity—we not aware Estimate the proportion of ready-to-hatch eggsinthe batchby checking their developmental stage. Ifthe volume of Check the condition of the eggsaftertransport. Open the package (eggsare normally shippedina small amount I I T ON D

eggs | I are capable of eating the entire size range of commercially theentire eating size of are range capable of URE Basic equipment needed for husbandry of CAL Bleach isastrong corrosive anditreleases toxic chlorine Fig. 7b

Mix 1 liter of water,Mix 1liter of kitchen 8–10g(one teaspoonful) of

STEP Mix 4, 8-10, NaCl 1lwater. 100and360gof each with  µ

CR , S/m Mix 1 ml of gentamicin sulfate with 999 ml of water. gentamicin 999mlof sulfate with Mix 1mlof c Do not allow the eggs to remain uncovered by the peat and exposed to low room humidity for longer I ), orwhitewhen dead ( T I 2

CAL ) ) can also be used, but fish may be more prone to Mix 0.002 g of methylene blue with 1 l of water. methylene 1lof Mix blue with 0.002gof

Hatched naupliimust available be atthe Mix 5 g of sodium thiosulfate with 1 l of water. 1lof sodiumthiosulfate with Mix 5gof

Fig. 5% Sodium hypochlorite5% Sodium solution (house

● House juvenile fish (from the age of 6

). Keep the filter in running the tank for T Artemia IMI N G eggs atleast24hbefore eggs 6–12h Artemia N. furzeri Fig. 7 eggs andstorage eggs d . ).

N. furzeri

-

ensure for afish thattheholeisnotlarge enough through.to get feeding. aholeatthelid’s Drill edgefor thefilter andheater air tube cord; lidto the plastic hole inthecenter allow for andconvenient easyhandling of lidisrecommended. thanaglass lidrather aplastic a5-cm useof The Drill ( aquaria neighboring thefish.condition of Coverwith alid to prevent tanks fish jumpinginto except you when are maintenance performing or checking thehealthand nearthewalls.tioned directly notusestrong Do above lighting theaquaria, (60 W), center-positioned bulb posi for aquaria a3×4mroom light with toonly limitmale–maleaggression. dimlighting For example, useastandard fish. 4lper volume of as Use thesametanksetup for juvenile fish. Provide Aquarium for setup adult fish filter outflow is not so strong that the current moves the fish involuntarily. Keep the bottom the of bare;aquarium provide no shelters. Ensure that the at least 3 weeks before adding fish, so that it is already colonized by bacteria. 2 5 6 Fig. 7 N. furzeri natureprotocols a ), opaquewithanadvanced

rarely jump but may do frightened). sowhen Housewith aminimum adult fishinatank 4 7 1

| VOL.11 NO.8VOL.11 3 8 protocol | 2016

9

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1401 - © 2016 Nature America, Inc. All rights reserved. embryos from diapause; these become ready tohatchin5–15d(at25°C). undeveloped eggsmay stillremain inthe peatafterother embryoshavehatched. The wetting often triggers dormant ( more important thanmaintaining high oxygen concentration overtime. Note thatincubation lengths are variable fill their swimbladder within the first5h.The increased oxygen concentration inthe water helps tominimizethe number of fishthat failto per 1literof hatching water. Fishbeginhatching within0.5–3hand perform vigorous ‘jerks’,withmost fishhatching hatched (depending onthe estimated number of eggs) or5hafterthe eggswere wetted, add one broken-up oxygen tablet 5| (Step 7 of the PROCEDURE) in the evening.  room temperature (25–27 °C). This normally takes 2–3 h.  swim bladders at one side of the aquarium ( next 3duntil the peatstopsfloating). Ensure thatthe bottom of the aquarium isslopedsothatthe waterleveliszero Stir any floating peatto release residual airthat may becausing ittofloat (repeat several times as required over the Fig. 4| maintain adepth of 2cmorshallower. can easilyswimoutand not get trapped inthe peat.Use alarger aquarium—or splitthe peatacross more aquaria—to to the volume of peat—the peatlayeronthe bottomshould beno deeper than2cmsothatthe freshly hatched fish 3| longer than 2 months after they reach the prehatching stage. sac begins diminishing ~60 d after an embryo reaches its pre-hatching stage gradually consuming their energy reserves. The precise time interval depends on the incubation temperature. The yolk  ready-to-hatch eggs required for experimental purposes.  damage them withthe tweezer tips. to transfer them inasmall amount of peatsoasnot to hatching). When moving eggswithtweezers, itissafer and proceed toStep3(the presence of peatpromotes eggs to50–100mlof boiledpeat(seeReagents section) entomological tweezerstogently move the ready-to-hatch hatched (e.g., for aspecific experimental purpose),usefine PROCEDURE. Ifonlyasmall subset of eggsare tobe a stock)are ready tohatch,proceed toStep3of the enough toobtainabreeding group of adult fishto maintain ready-to-hatch eggs. Ifenough (~50eggsshould be respective categories and estimate the proportion of can beseen( hatch, embryoswithconspicuous, gold-pigmented irises eyes ( (no eyeapparent; Most eggsthatare not yetready tohatchare clear looking for killifisheggsinthe peatcanbetime-consuming. gold-pigmented eye iris ( pigmented eyes ( to the naked eye ( ( Figure 7 1402 ?  a Figs. protocol

– TROUBLES

CR d CR CR CR CR ) Diapaused

8 Prepare oxygen tabletsbybreaking eachof them into 5–10smaller pieces. At the point when most eggshavealready Pour coldwater(15–16°C)into the aquarium. Keep the initial waterlevelat3cmabovethe peat(atthe deepest end; Pour outthe peatcontaining the eggsinto asmall (6l)emptyaquarium. Selectthe sizeof the aquarium according | I I I I I VOL.11 NO.8VOL.11 T Fig. 7 ). Immediately afterwetting, gently disturbany larger clumpsof peat(using fingers) tofree any trapped embryos. T T T T 2

I | I I I I CAL and Gross staging of CAL CAL CAL CAL

b STEP

H STEP STEP STEP STEP 5 ) canbeseen.When eggsare ready to Fig. 7 N. furzeri b 6 OOT ), and therefore itisuncommon for alleggsinabatchtodevelop synchronously; avariable proportion of a ); egg with a fully developed, ready-to-hatch embryo with 2 ); advanced, postdiapause II embryo with black- , they show astrong drive toreach the surface. Do not postpone the application of the oxygen tablets. Late application (>24 h after hatching) is largely ineffective. | 2016 Wet the eggs at midday to allow for oxygen treatment (Step 5 of the PROCEDURE) and first feeding Low water temperature stimulates hatching; however, the water should be allowed to naturally reach Ready-to-hatch Hatching success may vary greatly in I Fig. 7 N c c G ). Count the number of eggsinthe egg lacking structures that are readily visible ); and dead, decaying egg ( N. furzeri 6 | 2

. Breaking the tabletinto pieces increases oxygen concentration atasingle time point, which is natureprotocols a ), orsometimes black-pigmented eggs incubated in peat. Fig. 8 Nothobranchius ). Although itisunclear whether newborn killifish need togulpairinorder tofill their d ). Scale bar, 1 mm.

spp. embryos remain viable for only a limited time, as they are

N. N. furzeri

. . We suggest picking out approximately twice the number of c a 1 6 . . Do not continue to incubate eggs for d b

16,1 7

© 2016 Nature America, Inc. All rights reserved. Fish wean off the BBS and accept the new diet more easily when they are not distracted by other food items. Bloodworms are and use a Pasteur pipette for feeding to the fish. Introduce the bloodworms first and wait for ~20 min before adding the BBS. or a box knife to cut 2- to 4-mm-wide slices directly from a deeply frozen block. Melt the slices in a small amount of water smaller pieces for the first 4–5 d of bloodworm feeding, as full-sized bloodworms are still too large for the fish. Use a scalpel 10| reduces osmotic shock and thus facilitates longer BBS survival in the aquarium, prolonging their availability to the fish. To partly compensate for the decreased food delivery rate, add 16 g (a spoonful) of salt per N.10 furzeri l of water; the increased salinity siphoning out the waste from the bottom (use tubing with a 9-mm diameter). Continue feeding three times per day (juvenile 9| bag of eggs into an incubator (Step 16A of the PROCEDURE) and repeat wettingof the(Step PROCEDURE.3 of theCheck PROCEDURE)the re-driedafter 2–3 peat,weeks. as described in Step 2 of the PROCEDURE. If unhatched eggs are abundant, place the glass pipeorbyrepeating the pouring procedure. hatching aquarium veryturbid when disturbed).Check for any remaining fish,and transfer them tothe new tankusing a hatching aquarium withwaterfrom the new tankand waituntil the peatresettles (the fine peat may make the waterinthe remains atthe bottomof the hatching tank).Ifyouare indoubt whether alljuvenilefishwere transferred, refill the hatching aquarium tothe new aquarium. Donot transfer the peattothe new aquarium (itwillhavesunkbynow and 8| timing of the wetting. Overdue fish are very thin with no apparent yolk sac remnants. the oxygen tablet treatment. The appearance of the freshly hatched fish is a retrospective indication of the appropriate  growth rate onstandard BBS of fatty-acid-enriched BBS (e.g., SELCObyArtemia Systems) isnot required, asjuvenile 1 hafterfeeding no more than100–200livenauplii should remain; these tend toaggregate atthe brightest spot.The use quantity of BBStobeuseddepends onthe number of juveniles;initially, fishshould beliterally ‘swimming in food,’ but coating thatemerges asaconsequence of decayed BBSand thattypically occursatthe brightest spotof the tank.The exact yellow-colored bellies ~15minaftereachfeeding asasign of fullsatiation. With anairtube, siphon outthe substrate midday and lateafternoon). Distributethe BBSevenly across the hatching aquarium. Ensure thatallfishhavefull, 7| aquarium maintains a slightly acidic environment, keeping waste nitrogenous substances in a safer, ammonium form (NH overly disturbed and the fish are not buried. For the first 3–4 d, a 6-liter tank can safely house 200–300 juveniles. Peat in the the water surface covered by a plastic bag loosely floating on the water surface) so that the substrate on the bottom is not slow-flowing air tube as a hose or pouring the water onto Gently pourwater(e.g., using asmall-diameter, this means raising the waterlevelby3–4cmdaily). water eachday (e.g., ina6-laquarium (25×16cm), quality, add 100%of the original (wetting) volume of to the bottomof the aquarium. To maintain adequate water 3–4 times daily sothatitreleases residual airand settles aquarium heater. Continue stirring the floating peat tank inaheated room orusing asmall (25W)submersible maintain watertemperature at25–27°Cbyplacing the water ( use aglasspipetocatchthe fishwhile keeping them in If earlymanipulation isrequired for experimental purposes, sloped tanktoanormal, flatposition ~24hafterhatching. to avoid manipulation of still-fragile fish.Re-arrange the together withthe incubation peatfor 3–4dposthatching 6| R more nutritious than BBS, and therefore growth rates of aising aising juvenile fish

CR CR At ~10 d of age, begin supplementing the BBS diet with frozen bloodworms at each feeding. Chop the bloodworms into Change 30% of the water (ensure that the drop in water temperature is <5 °C after the change) three times a week while At the age of 3–4d, move the fishtoalarger aquarium (seeEquipment Setup).Slowlypouroutallthe waterfrom the For optimal growth, feedthe young fishwith Keep newly hatched fishintheir hatching aquarium I I T T I Fig. I CAL CAL should constantly have full, rounded bellies). For 5-d-old fish, two feedings per day is acceptable but suboptimal.

6

STEP STEP ). Donot useany aeration orfiltration, and After the juveniles are moved to a larger tank, dry the peat from the hatching aquarium, as described in Step 16A If the eggs were overdue, feed fish as soon as possible—at the start of swimming, in conjunction with ●

T IMI 1 8 N . G 3–4 weeks

Artemia

N. furzeri

nauplii (BBS)three times aday atregular intervals (morning, the other end. hatching aquarium with zero water level at one end and 3-cm water level at Figure 8 have the potential to increase after weaning.

| Setup for hatching peat-incubated natureprotocols N. furzeri canachieve optimum N. furzeri

| VOL.11 NO.8VOL.11 eggs. Sloped protocol | 2016

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+

1403

).

© 2016 Nature America, Inc. All rights reserved. shrimp, zooplanktonoranimal-protein-based fishpellets(e.g., SAK 55or SAK Energy byExot Hobby). When choosing an food from apathogen-free source tomaximize fishcondition. Occasionally, supplement the diet withliveand frozen brine conditions, the exclusive useof frozen, cultured bloodworms (Hikari) isrecommended. Forbreeding, feedthe fishwithlive diet—the fishconsume bloodworms willingly whileproducing decent clutches of 20–40eggs. Forstandardized experimental 14| the tanks covered (see Equipment Setup) and try to avoid placing tanks with different strains adjacent to one another. room. Keep all tanks clearly labeled with strain ID, date of birth and number of male and female fish. As a precaution, keep  with the leastdamaged fins) incaselethalinteractions are repeated. Remove the aggressive individual(s) from the aquarium. male–male interactions do occur. Try toidentify the particularly aggressive individual bydirect observation (often the male biased sex ratios inthe aquarium (male fish relentlessly courtfemale fish). Notethatoccasional nonlethal(orevenlethal) Maintain atemperature range of 27–32°C.Prevent excessive harassment of female fishby maintaining equal orfemale- and water exchange regime as for advanced juveniles. Alternatively, house the13| fish individually in a recirculating system ( C mature by4weeksof age under proper conditions. later ( Initially, male fishare recognized bydistinctly larger dorsal and anal fins, with signs of malecoloration developing 1–3d of secondary sexual characteristics appearat12–25dof age; sexual maturity canbereached atthe age of 18d(ref. other male and female fish(the rapidly maturing male fishcanbe returned to the same aquarium later). The first signs 12| ? with the separated individuals until they are capableof ingesting full-sizebloodworms (1–7d). of food shortage; itregularly occursdespite ideal feeding conditions. Continue the mixed BBS/chopped bloodworm diet incapable of consuming full-sizebloodworms atthisage. Thisisaninherent interindividual variability rather thanasign aggressive larger siblings during maturation. Some portion (5–10%)of the cohort may show stunted growth and be (adult diet) atthe age of 15d. Separate any stunted individuals and placeinanother aquarium toavoid injuries from 11| 1404 protocol are of the adult fish

weeks tomonths. wall) and eventually die. Some female fishcan release the eggseven without a male, but the initial capability is often lostwithin to spawnregularly, some individuals (30–80%)become eggbound (extremely swollenbelly, eggsvisiblethrough the abdominal  6. Collectthe eggsfrom the fishusing the pair-spawning method (Step15B of the PROCEDURE)twice aweek. every 3–4weeks(follow the manufacturer’s recommendations). of the tankstwice perweek(unlessthe recirculating systemisdesigned for automatic wastecollection). Cleanfilter media once but do not keep the lights onpermanently. Change 30%of the waterand siphon outorganic debris accumulated onthe bottom 5. Maintain anormal feeding regime (twice perday). Turn onthe lighting during the feeding events tocheck the condition of fish BBS wouldbefiltered out). Use ascreen (fryscreen) toprevent the small fishbeing washed down the filtersystem. 4. Introduce the fish. Water filtration canbeturned on when the fish reach the age of10–15d (the onset ofbloodworm feeding; 3. Measure the temperature of the waterand useapowerful,thermostat-equipped heater (300W)ifnecessary. Turn the UVlampon. for atleast3weeks. 2. Fillthe systemwithwater. Turn onthe waterpumpand allowabacterial community todevelop inthe filter by keeping itrunning single tankisatleast2land the waterinflowisupto0.6l/minorcurrent isbroken down byascreen. 1. Prepare arecirculating system.Commercially availablesystems for zebrafish are generally suitable, provided thatthe volume of a requirements for fishcare, including the labor of eggcollection and the need for more sophisticated equipment. (only tospawning periods); and (iv)limiting the potential for spread of disease(provided thatanUVlampisused). social environment ongrowth rate, maturation and lifespan(independent replicates); (iii)limiting the riskof intraspecific aggression housing. The advantages of individual housing include the following: (i)identification of individual fish;(ii)limiting the effects of For their entire lifespan, ● Box 1 TROUBLES

CR

Feedfishtwice per day (morning and evening) to obtain high reproductive output. Use frozen bloodworms asastaple Keep adult fish density ata maximum of 4l of waterperfish, and use the same aquarium setup(see Equipment Setup) Separate the most rapidly maturing male fishinto adifferent aquarium to facilitate the growth and maturation of the Assuming thatthe previous feeding regime and aquarium conditions were optimal, beginfeeding full-sizebloodworms The disadvantages of individual housing include the following: (i)imposing differences from natural conditions; and (ii)increasing CR

| I T VOL.11 NO.8VOL.11 Fig. 1 T I IMI T I CAL I CAL | N d

H

Individualhousingof STEP STEP ). Mature female fishhavebellies swollenwitheggs( OOT G 3–9months | 2016

Beware of strain contamination in cases in which several I N. furzeri N G | ● natureprotocols N. furzeri

T female fishcontinuously produce relatively large egg masses ifthey are fed well.Ifthey are not allowed IMI N G canbehoused individually insmall tanks inarecirculating systemasanalternative togroup 3–9 months N. furzeri inarecirculating system Fig. 1 c ) (from ~25mmlength). Most fishbecome sexually N. N. furzeri strains are maintained in the same

1

B

8

ox

).

1 ).

© 2016 Nature America, Inc. All rights reserved. ( ( a single pair and is used mainly for experimental purposes; this approach offers instant feedback andpairs high but lackscontrol direct over feedback clutches. regarding clutch size. By contrast, pair spawning requires the immediateis normally collection used for of general eggs from maintenance of 15| B to prevent deterioration of waterquality. A rounded bellyisareliable sign of satiation. Siphon outany uneaten, nonlive food from the tankupto2hafterfeeding dried food (pelletsand flakes) cannot bethe main component of alternative locallyavailable brand, keep inmind thatred orreddish food coloration isanimportant attractant. Note that B A reeding a Pasteur pipette( accustomed to the setupand spawn readily. To recover the eggsfrom the mesh-equipped aquarium, simplysiphon them outusing 3. When the mesh method isused, the initial 1–3spawnings may not yield the usual number of eggs, butthe fishsoonbecome the spawned eggsoutof the reach of the spawning pair. 2. Insert thisbox into another box of the same size, and allowagapof 15–20 mmbetweenthe two bottoms ( bottom of aplastic food box and replace itwithmesh. 1. Prepare the spawning tankusing a4–5-mm mesh toseparate the bottomof the container from the spawning fish.Cut off the entire (iii) (iii) (iv) (iv) (vi) Box 2 (ii) (ii) ) ) Group spawningmethod (v) (i) (i) To obtain eggs, breed the fish in groups (option A) or in pairs (option B). Group spawning is a long-term approach that P air spawningmethod cool down toroom temperature. tank; avoid dropping food into the spawning jarduring feeding. during group spawning); asparticular fishtend to specialize incannibalizing eggs, remove these individuals from the conceal the eggs;keep the fishconstantly satiated (continual presence of live food inthe tankisespecially beneficial as aspawning substrate. Alternatively, useanaquarium setupwithout the substrate ( Prepare alid tolooselycoverthe container. Use a0.5-to1-cmlayerof laboratory glassbeads(seeReagents section) to prevent fishspawning outside the jar. at leastsome peatfrom the jar, which isareliable sign thatspawning hascommenced. Siphon outany spilledpeat 15 female fish for an80-litertank).Fishwillbegintoenter and spawninthe jarwithin1–2 d. Normally, fisheject maintaining maximum adult fish density (4lperfish). Keep the sex ratio at1:3 (male:female—e.g., 5 male and at least10cmof waterabove the jartoenable free movement of the fish.Introduce agroup of parental fishwhile spawning jarinside the aquarium veryslowlysothatthe waterdisturbance does not washthe peatout.Maintain jars withanopening of atleast8cmindiameter ( boiled peat;peatprotects the eggsbetterthanglassbeadsduring long-term exposure toadult fish. Use bottle-shaped blue solution (0.002 g/l). filled with water for sieving the eggs from the spawning substrate, an empty Petri dish to hold the eggs and methylene Wait for at least 5 h (preferably 12 h) before recovering the spawned eggs (the egg envelope hardens). Prepare a vessel immediately afterbeing paired.  for laying allthe eggsthatthe female fishhasovulated. Returnthe fishto their home aquaria afterspawning. spawning container. Keep the pairinthe spawning container for 2h(no aeration needed)—this isnormally sufficient PROCEDURE every2weeks. male fishtoenable breeding of subdominant male fish.Change and dry the peatas describedinStep16A of the Meanwhile, prepare asmall (2l,15×10cm)aquarium ortub(clearplastic food boxes workwell; Separate female fishfrom male fish for48hto maximize egg yield. Swap individual male fish inthe aquarium every24horadd anadditional spawning jarpereachadditional three  Add aquarium watertocompletelyfillthe jarand wait for the peattosettlethe bottom(5–10min).Placethe Prepare spawning placefor fishbyfilling aglassjar(15-cm height, minimum volume 0.6l)witha6-cmlayer ofwet After removing the fish,briefly check whether the pairactually spawned: hold the tankagainst a light source, placean Introduce asingle male and female into the tub. Maintain dimlighting and minimizeactivityinthe vicinity. Coverthe Fill the spawning container withwater. Use warmwater(maximum 34°C)for the startof spawning butletitnaturally N. furzeri container. Afew(1–4)eggsshould become dislodged from the substrate. index finger into the spawning substrate and move itslowlyalong the fulllength of the shorter side of the spawning

CR I | T Pair spawning of I CAL tend tocannibalizetheir eggs. To minimizecannibalization, maintain an~6-cm-thick peatlayertobetter Fig. 4

STEP g Pair ) (cutthe tip off). ● N. furzeri

● T IMI

T IMI N G N regularly (every2–4d)tohabituate them tohandling and tostartspawning 3.5d G 14d N. furzeri N. furzeri cultures. It requires only periodic egg collection from multiple spawning withoutsubstrate Fig. 6 ). The bottleshapeprevents excessive spilling of the peat. N. furzeri diet, asthe fishonly reluctantly eat dried food. ● natureprotocols

T IMI B ox N 2 G ). 2h Fig. 4

| VOL.11 NO.8VOL.11 c ). Thisholds Fig. protocol | 6 2016 ).

|

1405

© 2016 Nature America, Inc. All rights reserved. ( manipulation of the embryos. a high degree of control and observation of developmental processes indetail, and offers atool for potential chemical observation of development during anexperiment. Incubation inwater(oramodified aquatic medium) allows for both natural conditions. Incubation ontopof peatincorporates dry (natural) incubation whilesimultaneously allowing for gross experiments not requiring continuous feedbackonactual embryonic development. Thismethod is most closelyrelated to of peat(option C)orinwater(option D).Incubation inpeatisusedfor general maintenance of 16| Incubation of eggs 1406 (viii) protocol A (vii) (iii) (iv) (ix) (ii) ) Incubationof group-spawned eggs (x) Incubate the fertilizedeggsasdescribed inStep16B–Dof the PROCEDURE. (i) To incubate group-spawned eggsinpeat,follow option A.Incubate pair-spawned eggsinpeat(option B),ontop

| VOL.11 NO.8VOL.11 the package infront of afan (shortens waiting to~1/5of time). (i.e., drier thanrest of the peatball).Ifthe peatisstilltoodamp, rewrap itand waitfor anadditional day orplace hue (otherwise itistoodry); the peatsurface directly adjacent tothe filterpapershould bejuststarting toturnpale no signs of waterreflectance inside the peatgrain structure (otherwise itistoowet)and willhaveavery dark brown the surface of the filterpaperis dry; thisusually takes 2–4 d. immediately withthe strain IDand date of collection using apermanent marker. Leaveitatroom temperature until ? methylene bluesolution. a Pasteur pipette( water from the Petri dishbysiphoning itoutwith female sizeand condition. Remove the washing Expect ~20–120eggsperfemale fish depending on remaining eggsfrom the sieve into alarge Petri dish. free of hard particles (seeReagent Setup). until the waterfrom the peatstopsdripping. The eggswillnot bedamaged aslong asthe peatisfine structured and Step 8of the PROCEDURE)from the tankand pourallthe peatwitheggsinto the net. Squeezethe net firmlybyhand used tosieve BBS).Remove the spawning jar(orpreviously wettedpeatpotentially containing undeveloped eggsfrom the sieve (eggsare clearlyvisibletothe naked eye). the sieve. Check whether there are any eggscaught in circular movements towashthe substrate awayfrom does not spilloverthe edges of the sieve. Use slow, the sieve inthe watervesselsothatthe substrate particle rubbing against the eggsurface). Submerge the substrate tothe seive(thisminimizessubstrate Use the watercurrent from awashbottletotransfer substrate bysieving the substrate through the mesh. mm) ( strainer, asthe mean sizeof ? spawning (Step 15A),taking appropriate corrective measures. If no eggsare visibleafter 30sof searching bystirring the peatrepeatedly, spawning wasunsuccessful. Repeat If the spawning issuccessful, meaning that~5eggs are visible(eggsare plentiful), proceed tothe next step. pipette. The fertilization rate isvariable, butthe typical range is70–90%. start decaying and acquire abluish,rather opaque coloration ( and the vitelline membrane) and remain clear( illumination. Fertilizedeggshaveadouble-layered structure withaperivitelline space(alayerbetweenthe chorion in water;seeStep16Dof the PROCEDURE).Check the fertilization rate under astereomicroscope withbottom ~12 h(or3dwhen fertilizedeggswillbeincubated Keep the eggsinthe methylene bluesolution for Use alaboratory washbottle( Use asieve withamesh sizeof ~1mm(e.g., atea Check for the presence of eggsinthe peat.Separate the clump(the usual volume is150–250ml)into halves. Unwrap the package tocheck the dampness of the peat.Ifthe peathas the correct moisture content, itwillshow Empty the contents of the hand net onto the filterpaperand wrap itupto form asolid package. Labelthe package Prepare double-layered sheets of filterpaper(or newspaper). Prepare a net withfine meshsize (e.g., a0.15-mm mesh

TROUBLES TROUBLES Fig. | 6 2016 ) torecover eggsfrom the spawning H H OOT ● OOT

| T natureprotocols IMI I I Fig. N N G G N G 6 17 d to 16 months ), and replace itwiththe

N. furzeri Fig. 6 ) torinse out eggis1.35 Fig. 9

a ). Bycontrast, after12h,the single-layered, unfertilized eggs unfertilized ( Figure 9 a a ) A fertilized

Fig. 9

| Differences between a fertilized and an unfertilized N. furzeri b ). Remove unfertilizedeggsusing aPasteur N. furzeri egg lacking perivitelline space. Scale bar, 0.5 mm. egg with a typical double-layered structure. ( b N. furzeri culture and in N. furzeri

b

) An egg.

© 2016 Nature America, Inc. All rights reserved. (D) Incubationof pair-spawnedeggsinwater ( ( (viii) Before hatching, check the developmental statusof the eggsinside the bag,asdescribed inStep2of the PROCEDURE. C B (vii) (iii) (iii) (iv) (vi) ) Incubationof pair-spawnedeggsontopof peat (ii) (ii) (ii) ) Incubationof pair-spawnedeggsinpeat (v) (i) (i) (i) plastic zipperbag(several Petri dishes canbeplacedinthe same bag)toprevent rapid evaporation. Step 16A(vi—viii)of the PROCEDURE. the next 1–3d. When the correct moisture levelisachieved, sealthe bagand continue incubation asdescribed in the moisture levelisonlyslightly higher thanisappropriate. Check the dampness of the peatrepeatedly over of the bagthoroughly until the correct moisture levelisachieved. Leavethe bagopenand placeitinthe incubator if wait for another hour ifitistoodry, repeating asrequired. Ifitistoowet,add some dry peatand mixthe contents bleach solution for 5min(useatimer). partly removes fibrilscovering the egg surface and makes the embryo more easilyobservable. Keep the eggsinthe water. Whilegently stirring the water, add 1mlof the bleach;the bleachnot onlysterilizesthe eggsurface butalso can bepooledifallowedbythe experimental design). Pour off the methylene bluesolution. Add 200mlof sterilized with aminimum volume of 200ml.Transfer the eggstogether withthe methylene bluesolution to the vial (clutches top of the peat,asingle egg percell. Flip the bagupand down and stirthe peattoavoid clumping. peat. Ensure thatthe eggsare evenlydistributedwithinthe peattolimitthe potential spread of any fungal infection. the bag.Transfer eachegg withaslittlefluid aspossible (e.g., transfer the eggsingroups) toavoid overwetting the incubation, becausesome fluid willbetransferred withthe eggs. Transfer the peattoaplastic zipperbag. container orbywrapping itinfilterpaper);should haveaslightly lower degree of moisture than required for entire volume of the solution daily bysiphoning and replacing it. the middle of the cell. of the PROCEDURE).Press the peatwithfingers so thatitssurface isfirm; create aconcave shapeto keep the eggin mix insome new dry peattodecrease the overall moisture level. upside down afteradding the water, and waitfor ~1htoallowevensoaking of the peat.Iftoomuch waterisadded, starting todry outand afewdrops of watershould beadded; mixthe peatthoroughly byflipping and turning the bag proportion of bellysliders, atleastinsome laboratories. > peat increases developmental speedcompared withincubation inside the peat(especially atincubation temperatures newborn fish often sufferfrom bellysliding (inability tofill the swimbladder and swim normally). ensue: the time window for successful hatching shortens considerably; the embryosexhibit hatching problems; and  (For anoverview of husbandry stepsand incubation approaches, see development. Expectthe eggstobeready tohatchwithinanadditional 4–6weeks(i.e., 8–10weeksintotal). the temperature to25–26°Cinduce most embryostoexit diapause simultaneously and achieve synchronized To induce dormancy inmost eggs, incubate the eggsat17–20°Cfor atleast4weeksaftercollection; switch to 22–25°Cachieve erratic development; eggsthatenter adiapause willdevelop unevenly for 2–16months. (nondiapausing) embryos;eggsthatskipalldiapauses are ready tohatchwithin17–21d. Setthe temperature Set the incubation temperature to27–28°Cspeedupdevelopment and toincrease the proportion of escape (17–28 °C).Keep inmind thatthe effectof incubation temperature isnot fullydeterministic (seeINTRODUCTION). Wait for 1haftertransferring the eggsbefore checking the dampness of the peat.Add afewdrops of waterand Use aPasteur pipettetotransfer the eggsfrom the methylene bluesolution (from Step15B(ix)of the PROCEDURE)to Prepare 200–300mlof peat(seeReagents section). Allowittodry until apalebrown hue isreached (inanopen Check the moisture levelinthe bagsperiodically every1–2months. Ifthe peatislosing itsdark brown hue, itis Place the baginanincubator. Setthe incubation temperature according tothe desired speedof embryodevelopment Using aspoon,move the peatwitheggsinto aplastic zipperbagand sealthe bag.Using apermanent marker, mark On the fourth day afterfertilization, prepare 5%sodiumhypochlorite (household bleach),sterilizedwater and avial Incubate the fertilizedeggsinmethylene bluesolution (from Step15B(ix)of the PROCEDURE)for 3d, changing the Start incubating the eggs asdescribed inStep16A(vi–viii) of the PROCEDURE.Note thatincubation ontopof the Cover the dishwithalid. Mark the lid withdate and strain ID.Sealthe lid withParafilm orplacethe dishinto alarge Use aPasteur pipetteto transfer the eggsfrom the methylene bluesolution (from Step15B(ix)).Placethe eggson Prepare acompartmented Petri dish( embryo survivallater. with descriptive information oneggdensity (e.g., high, intermediate and low)toenable feedbackonapproximate the bagwithimportant details (the date of batchcollection, strain ID).It ispractical toadditionally labelthe bag 25 °C).However, eggsincubated onthe peatsurface often hatchpoorly, and thisapproach results inalarger

CR I T I CAL

STEP Note thattemperatures higher than28°Cleadtosuccessful embryodevelopment Fig. 6 ) and filleachcellhalfwaywithpeat of the correct dampness (Step16A(iii) Fig. 5 .) natureprotocols

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© 2016 Nature America, Inc. All rights reserved. this this would pose an additional stress load for fish in a new environment. solution) methylene blue, malachite green or broad spectrum antibiotics) without observing symptoms of a real disease, as  facility becauseof the complex lifecycleof boththe fishand their parasites. fish are often heavily parasitized, but many metazoan parasites pose no threat of adirect infection toother fishin the time line of the development of any potential problem. Consult withaspecialist ifany problems are suspected;wild-caught a protocol withdate, time and results of the observation; describe the appearance of the fishand their behavior toobtaina 23| different persontotake care of the quarantined fishthan for established stocks, ifpossible. established stocks. Avoid indirect contact through, e.g., waterhoses, waterbuckets, splashing waterorwethands. Assign a isolated tank.Use onlydedicated equipment for any manipulation. Donot allowany direct contact of the new fishwiththe 22| Quarantine procedure for new fish and eggs Low temperatures may kill the embryos.  ! 21| the sealedbag.Labelthe bagwithstrain ID,date of the spawning and number of eggs. 20| eggs. Adjust the final dampness of the peatbyadding some dry peatif necessary (Step16A(iii) of the PROCEDURE). eggs into the bagwithpeat.Avoid making the shipping peattoowetbylimiting the amount of watertransferred withthe 19| incubated, ifpossible. 18| sensitivity tohandling stress and adverseshipping conditions thanthe resistant diapause IIeggs. diapause IIorready tohatch).Donot useprediapause II(<9dpostfertilization) eggsfor shipping becauseof higher 17| S

1408 (viii) protocol

hipping hipping eggs (vii) (iii) CAUT (ix) (vi) (iv) (xi)

(x) (v) CR CR Observethe behavior and appearance of the fish for atleast10mineach day (assign an experienced person).Prepare Keep any new fishcoming tothe husbandry facility quarantined for 30 d. Placethe fish into adisinfected, completely Wrap the baginbubblewrap and placeitinto anenvelope. Shipthe eggs. Sealthe bag.Prevent clumping of the eggsand achieve evendistribution of the moisture byflipping and shaking Use entomological tweezerstomove dry incubated eggsinto the bag.Use aPasteur pipettetomove water-incubated Prepare asmall (10×6cm)plastic zipperbagwith20mlof peat.Use the peat inwhich the eggswere originally Selectsuitableeggsfrom the eggstock;useonlyeggsthatare atleast3weekspostfertilization (presumably in

| I I VOL.11 NO.8VOL.11 the medium. Placethe dishinto the incubator at25°C. Pasteur pipette. The medium levelshould be~1cm.Sealthe dishwithParafilm toprevent rapid evaporation of T T incubation period and handled antiseptically thereafter fungus thanduring incubation inthe peat.They must betreated withantimicrobials atthe beginning of the  normally). Incubate the embryos as described for pair-spawned eggs in peat (Step 16A(vi–viii) of the PROCEDURE). Transfer the diapause II or postdiapause II embryos to damp peat (this transfer is important for are prominent and 32somitesare visible)oramore advanced stage (some dark orgolden pigmentation inthe eyes). Wait for the embryostoreach diapause II(ref. of sterilizedwater. Gently stirand waitfor ~1min. Trigger the exit from diapause IIbyincreasing the temperature to28–30°Cfor 48h(the embryos develop tothe Using astereomicroscope, check the developmental statusof the eggsonaregular basis(e.g., 2times perweek). Distribute the eggswiththe solution into compartmented plastic Petri dishes, one eggpercell,using asterile Pour off the waterand add 200mlof 10mg/l gentamicin sulfate solution Pour off the watercontaining the thiosulfate solution, using aPasteur pipettetoremove itcompletely. Add 200ml Pour off the bleachsolution, taking care toremove itcompletelyusing aPasteur pipette. Add 200mlof While gently stirring the water, add 1mlof the bleach.Wait for 5min(useatimer). Pour off the bleachsolution. Add 200mlof sterilizedwaterand stirgently. Wait for 1min. prehatching stage withinthe next 8–10difthe exit wastriggered successfully). (use the timer). sterilized water. Add 1mlof sodiumthiosulfate solution (5g/l)toneutralize any remaining bleach.Wait for 5min I I I ON CAL CAL

CR For international shipping, follow applicable laws and regulations. I

T STEP STEP I CAL ● |

2016 Do not apply any unspecific chemical treatment (e.g., acriflavine, FMC (formaldehyde–malachite green Do not send the eggs if a temperature above 4 °C cannot be guaranteed during the shipment. T

IMI STEP | N natureprotocols During incubation inwater, G 1–2 h ●

T IMI N. furzeri 1 N 6 G ) (atthe age of 9–11dpostfertilization, optic lobesand optic cups 30 d 6 eggsare more prone toinfection withbacteria and/or 3 . 6 3 .

N. furzeri eggs to hatch

© 2016 Nature America, Inc. All rights reserved. ( present a high toxicity risk. Never use copper sulfate, as its toxicity is especially high for  of experimental fish.  mortality). high suffer eggs spawning, cease (fish treatment the during fish the of reproduction the stops and aquaria other to disease the spreading of risk the increases treatment However,long-term the limited. is space if used be should option this so aquarium, infected the in directly treated be can fish B), (option bath long-term the performing When bath. long-term B, option or bath, short-term A, option use Tooodinosis, treat infected. already are tank the in fish all recognized, are disease the of symptoms When infection. of stage final very the until eating continue fish ( water hard with <300 (conductivity water soft in common are more outbreaks Oodinosis are folded. fins the later but spread, fins their keep fish stages, early the in symptoms: Behavioral fish. the behind source light the with them at looking when dotted be to appear fish female the of fins Transparent fin. dorsal the of front in dorsum the of part proximal very the on and (eyebrows) eyes the above concentrated more are but surface body whole the ( surface fish’s the on spots yellowish distinct but mm) (0.1 small very for search symptoms: visually. Morphological it inspect and aquarium 25| O the parental fishare proven tobe healthy. Maintain everyday preventive quarantine ( handling stress. Incubate the collectedeggs(Step 16Aof the PROCEDURE),buthatchthem tofound anew strain onlyif 24| A odinosis odinosis treatment contamination. 4. Boil(orautoclave)spawning and incubation substrates aftereachusetoremove potential pathogens and toprevent strain if possible. Minimizethe extent of wetting hands during manipulation intanks whenever possible. and pipettestominimizethe riskof pathogen transfer betweentanks. Use adedicated siphoning/water change hose for eachtank, 3. Avoid contact of tankwaterwiththe waterfilling hose (keep itoutside the tankwater during filling), feeding scoops, tweezers stable thanbleach. maintain some undissolved saltonthe bottomof the vessel.The useof saltposesno threat tothe fish,and the solution is more after use. Ensure thatthe solution remains saturated (some amount of saltislostwitheachuseof the hand net) byadding saltto a vesseland fillitwiththe saltsolution. Keep allhand nets constantly wholly submerged inthe solution, and return each of them of saltper1lof water. Use the solution for disinfecting the manipulation equipment such ashand nets. Equipthe fish room with 2. Use onlydisinfectedequipment for any manipulations of the fish.Prepare saturated NaCl (kitchen salt)solution byadding 360g Ensure thatthe waterisfree from chlorine using achlorine teststrip. Return the filterand turniton. Add 10ml of thiosulfate solution per100ls of water. Wait for atleast1h.Emptyand refill the tank. it thoroughly under running waterbysqueezing itrepeatedly for atleast5min.Emptythe tankcompletelyand refill itwithwater. per 100lof waterdirectly tothe tank.Keep the filterinthe tankand running. Wait for atleast30min.Remove the filter and wash Disinfect eachtankwithsodiumhypochlorite solution before introducing any different group of fish. Add 10ml of sodium hypochlorite 1. Keep tankswiththe fishisolatedfrom eachother, asthe use of liveand frozen foods presents a risk of infection withapathogen. (iii)

Box 3 (ii) ) CR CR (i) Keep collecting eggsusing the group-spawning method (Step15Aof the PROCEDURE),which results inthe least If oodinosis is suspected, examine a live individual fish under a strong external lamp. Place the fish in a small a small in fish the Place lamp. external strong a under fish individual live a examine suspected, is oodinosis If T I I reating oodinosiswithashort-termbath aquarium, and another for removing fishfrom the infectedaquarium.  second hand net. after transfer). Wait for another 5sand then quickly transfer allmotionless fishtothe new aquarium, using a for the fishtoswimin normally, sothey willlie ontheir sides after5–10 s. solution ingroups of 3–5individuals. The fishwill become veryagitatedat first. The saltsolution istooconcentrated aquarium tothe second plastic tub. Using one of the hand nets, beginplacing the infectedfishinto the 10%salt small (2-l)disposableplastic tubs. infection-free tank;twodisinfectedhand nets (submerged insaturated saltsolution for atleast1h);and two T T Observe the fish. Wait until their opercular opening (breathing) stopsand they remain motionless (~10–30s Pour the 10%saltsolution into one of the plastic tubstoawaterdepth of 2cm.Transfer allfishfrom the infected Prepare a10%solution of NaCl and aquarium water(100gsaltper1lwater).Prepare anew, well-aerated, I I

CAL CAL CR I |

T STEP STEP Everyday preventive quarantine I CAL The use of remedies other than salt treatment to cure oodinosis is not recommended. Other remedies Note that treatments for disease may have longer-term effects that interfere with functional assessments >

500 500 STEP

µ Use twoseparate hand nets: one net for transferring fishfrom the saltsolution tothe clean S/cm Fig. 3 Fig. 2 ). Juvenile fish appear to be more resistant to the disease than are adults. Note that that Note adults. are than disease the to resistant more be to appear fish Juvenile ). b ), in contrast to the surface of a healthy fish ( fish healthy a of surface the to contrast in ), ●

T IMI N G 30min ●

T IMI N G 5–120min Fig. 3 Fig. B natureprotocols ox a µ Nothobranchius 3 ). These are found throughout throughout found are These ). S/cm ). 2 ) as compared compared as )

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© 2016 Nature America, Inc. All rights reserved. T Troubleshooting advice canbefound in ? 32| 10 ml/100lsodiumhypochlorite solution ( 31| inside the abdominal cavityonvarious organs ( 30| Glugea infections. treating in success ~80% in results treatment this that shows experience Our weeks). (~4 disintegrate naturally they 29| running. filter the Keep normally.fish the Feed activity. inhibitory antifungal and antibacterial with brown) water the (dyeing acids tannic ( almond sea large Addone infection. ( 28| T remaining fish. 27| of individuals from the stockshow the symptoms. upward ( 26| Dropsy treatment ( 1410 able able Fig. 3 Fig. 5 S protocol reatment of unspecified infections B

TROUBLES (iv) tep (ii) ) (i) Quarantine any new fishoreggscoming tothe laboratory. Discard allinfectedfish,their eggsand any peatusedtoincubate them. Thoroughly sterilizeallequipment using Lookfor parasitic xenomas todiagnose The condition isuntreatable. Discard the fishwiththe diseaseand follow recommendations for fishcare for the Observethe appearance and behavior of fishtodiagnose dropsy. The body is generally swollen,withscalesprotruding Infected fish have decreased activity and appetite, clamped, whitish fins and protruding eyes (exophthalmos) (exophthalmos) eyes protruding and fins whitish clamped, appetite, and activity decreased have fish Infected Return to the usual water change schedule after 3 d of salt treatment. Keep the leaves in the aquarium until until aquarium the in leaves the Keep treatment. salt of d 3 after schedule change water usual the to Return T

| reating oodinosiswithalong-term bath VOL.11 NO.8VOL.11 infection using saturated saltsolution (e.g., hand nets; see to the normal feeding regime. Keep the filtration on. is welltolerated) tothe aquarium. Maintain thisconcentration for 3dbyomitting allwaterchanges whileadhering 2 2 as treatment of infectedfish needs tobeperformed within days. amount of stress tothe fish,fish are temporarily distressed during treatment. Apply forethical approval inadvance, ! transfer, they willstartswimming soonand willfullyrecover. Maintain usual care afterthe treatment. that killsthe parasite. Although the fish may initially remain lying onthe bottomand breathing heavily afterthe should bevisiblyclearof spotsimmediately aftertreatment, asthe concentrated solution causesan osmotic shock Revert backtothe normal waterchange schedule after3d. Disinfectallequipment thatcame into contact withthe Perform a50%waterchange and then add 40gof looseNaCl per10lwater(waterconductivity upto7,000 Check the fishtreated withthe saltsolution under astrong light (e.g., 60-Wlight bulbabovethe tank). Treated fish c spp. treatment

CAUT | ). Use NaCl in the same manner as for long-term oodinosis treatment (Step 25B) to treat an unspecified unspecified an treat to 25B) (Step treatment oodinosis long-term for as manner same the in NaCl Use ). Fig. 3

Troubleshooting table. H Eggs Eggs do not hatch P I roblem OOT ON d ). Asick fishhasconsiderably decreased activityand stopseating. Typically, onlyasingle orsmall number | Applyfor ethical approval toperform the treatment. Although the procedure minimizesthe overall 2016 I N ● G

T IMI | natureprotocols ●

N T IMI G 5 min N substances) temperature and absence of humic Lack of stimuli (oxygen concentration, P G ossible reason 3 d T.cattapa ● T

able T IMI B Glugea ox N 2 G Fig. . 3 ) leaf per 20 l of water. After 1–2 d, the leaves will leach humic and humic leach will leaves the d, 1–2 water.of After l 20 per leaf ) ● 3 d ).

spp.These are manifested aswhitecystsupto5mmindiameter found T 4 IMI ). N G 3d

B

ox 3 overdue or damaged) should hatch (provided they are still viable, i.e., not Reagents section). Wait for 0.5–1 h. Most eggs (80–90%) layer of the glass beads used for pair spawning (see water to a depth of 2–3 cm. Cover the eggs with a 0.5-cm unhatched eggs to a vial that is 5–8 cm wide. Add hatching Forced hatching. Using a Pasteur pipette, transfer the S olution ).

(continued) µ

S/cm

2

© 2016 Nature America, Inc. All rights reserved. E. E. R to M.R. We thank M. Vrtílek and three anonymous referees for comments, Nothobranchius A stochastic nature of their embryonic development. from other fishlaboratory models—most notably the need for higher rations of high-quality food (liveorfrozen) and the in thisprotocol are followed. We reiterate thatthe successful husbandry of and histologically apparent. Juvenilemortality depends onrearing density butisnegligible (<10%)ifallmeasures described in ourfacility, wild-derived populations live4–8months and fishdie from senescent deterioration thatis macroscopically in female fishatthe age of 8weeks(after rapid growth ceases).Fishlongevity depends onthe laboratory population; reached in3–4weeks;fulleggproduction (40–80eggsper2hspawning sequence, repeated everysecond day) isachieved By using thisprotocol, weachieve hatching and proper juveniledevelopment in70–90%of embryos. Sexual maturity is ANT B B B Steps 30–32, Steps 28 and 29, treatment of unspecified infections: 3 d Steps 26 and 27, dropsy treatment: 5 min Step 25B, treating oodinosis with a long-term bath: 3 d Step 25A, treating oodinosis with a short-term bath: 30 min Steps 22–24, quarantine procedure for new fish and eggs: 30 d Steps 17–21, shipping eggs: 1–2 h Step 16, incubation of eggs: 17 d to 16 months Step 15B, pair spawning method: 3.5 d Step 15A, group spawning method: 14 d Steps 13 and 14, care of the adult fish: 3–9 months Steps 6–12, raising juvenile fish: 3–4 weeks Steps 1–5, hatching (shipped) eggs: 6–12 h ● T All All procedures described herein are in accordance with Czech legal regulations helpful comments, and R. Spence and S. White for correcting the English. able able and 15B(viii) 11 S 16A(iv) ckno ox ox ox

tep ˇ T ehulková for help with illustrations, A. Dorn for providing IMI I 3 2 1 C w , , everyday preventive quarantine: 5–120 min , pair spawning of , individual housing of I 2 2 le PATE N | dgm G

Troubleshooting table (continued). comes from the Czech Science Foundation (P506/11/0112) D ents production Low egg cannibalism Juvenile sliding Juvenile belly P Glugea roblem RESULTS

Financial support for our current research on spp. treatment: 3 d

N. N. furzeri

N. N. furzeri Insufficient feeding Malnutrition concentration Unknown/low ambient oxygen P without substrate: 2 h ossible reason in a recirculating system: 3–9 months Figure N. furzeri 4 and

are tolerant toawide range of waterchemistry.

photographic documentation. All authors contributed to the final text. last 10 years. M.R. initiated the paper. M.P. drafted the protocol. R.B. prepared AUT Biology, Czech Academy of Sciences. and have been approved by the ethical committee of the Institute of Vertebrate H OR

shrimp shrimp > frozen zooplankton > dry food) zooplankton > frozen glass worms > frozen fairy larvae > live glass worms > frozen bloodworms > live live (ranked from best to worst in terms of egg production: twice a day Increase the food ration for parental fish. Feed fish fish are fed properly Increase the food ration. Cannibalism is extremely rare if lethal to the fish dosage. An oxygen concentration that is too high can be in their swim bladders at a single time, which may further aid the fish in filling the chemical reaction and maximizes oxygen concentration Crushing the tablet further increases the surface area for Crush it into a fine powder. Add the powder to the water. Prepare one oxygen tablet per 1 l of hatching water. Increase the oxygen concentration in the water. S CONTR olution Tubifex N. furzeri I BUT I ONS worms > live bloodworms > live mosquito ad ad libitum

M.P., R.B. and M.R. developed the protocol over the stems from appreciating their difference natureprotocols and provide higher-quality food 6 2 . . Note: Do not increase the

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© 2016 Nature America, Inc. All rights reserved. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. c at online available is information permissions and Reprints interests. CO 1412 om/reprints/index.htm protocol

M

PET from the genome of a short-lived fish. short-lived a of genome the from (2015). 1539–1554 lifespan. of architecturegenetic and evolution into insights 133 fish annual the of brain the in levels. mRNA furzeri Nothobranchius short-livedfish (2012). 1133–1141 (species: killifish transgenicof aging. for model killifish African short-lived the in furzeri Rev. Biol. annual the fishes. annual African in timegeneration short killifish. annual an in hatching and diapause egg duringbet-hedging and plasticity phenotypicenvironment: fishes. annual of eggs the in duration variable of diapause embryonic and Pre-embryonic lifespan. shortest the with vertebrate killifish, African the of use habitat and furzeri fish short-lived the in capacity regenerative fin in decline resistance.stress and variables Drosophila mortality.extrinsic to response 3 fish annual short-lived the of strains research. furzeri Nothobranchius World the of Fishes 46 age-dependentneoplasias. ofincidence high by reinforced fish Gerontol. Exp. (1961). 146–149 (2012). 2175–2198 toxicants. to stages life early fish of responses the Lett. Ecol. disease. Rev.Res. Ageing Wit, J., Loeschcke, V. & Kellermann, V. Life span variation in 13 in variation span V.Life Kellermann, V.& Loeschcke, J., Wit, E.T.Tozzini, E. Terzibasi, aging for waters Fertile M. Kaeberlein, & Promislow,D.E. A.M., Wang, fish annual the in lifespan short Extremely A. Cellerino, & S. Valdesalici, Wildekamp,R.H. annual short-lived The A. Cellerino, & G. Rossi, E.T.,Tozzini, E., Cicco, Di Reichwald, K. Reichwald, D.R. Valenzano, M. Baumgart, A. Petzold, Inducibletransgenicthe C.expression inDuan, & H.Kamei, J.B.,Allard, generationthe for protocol microinjection A C. Englert, & N. Hartmann, Transposon-mediatedtransgenesisA. Brunet, & S. Sharp, D.R., Valenzano, model: teleostean emerging an of atlas Brain D’Angelo,L. bench: the to bush the From M. Reichard, & D.R. Valenzano, A., Cellerino, and maturation early growth, RapidPolacˇik, M. R., Reichard, Blažek, & M. variable a in Adaptation D.N. Reznick, & K. Lee, A.I., Furness, III. fishes. annual of biology developmental J.P.TheWourms, Polacˇik,M., Sedlácˇek,Reichard,polymorphism & colour M. Distribution, O. Age-dependentC. Englert, & B.Hoppe, N., Hartmann, Wendler,S., Egami, N. & Etoh, H. Life span data for the small fish, small the for data span Life H. Etoh, & N. Egami, female reproductionin and Age A. Comfort, understand to behaviour and physiology P.L.Using McNeil, & K.A. Sloman, speciation. and selection sexualEcology, O. Seehausen, & M.E. Maan, human modelling for species fish diverse zebrafish: the Beyond M. Schartl, research. aging for models as fish laboratory Small G.S. Gerhard,

, e3866 (2008). e3866 , | , 249–256 (2011). 249–256 , VOL.11 NO.8VOL.11 I , 226–233 (2012). 226–233 , N Nothobranchius furzeri Nothobranchius G G FI . . Anat. Rec. Anat. Cell Aging Dis. Model. Mech. Model. Dis.

Cell NANC

species: a comparative study on life span, environmental span, life on study comparative a species: 91 14 Nothobranchius et al. et , 511–533 (2016). 511–533 , et al. et et al. et

, 591–602 (2011). 591–602 ,

BMC Genomics BMC 160 et al. et et al. et 4 I Nothobranchiusfurzeri , 127–129 (1969). 127–129 ,

et al. et AL 6 A World of Killies: Atlas of the Oviparous CyprinodontiformOviparous the of AtlasKillies: of World A The transcript catalogue of the short-lived fish short-lived the of catalogue transcript The

G3 (Bethseda) G3 |

, 814–815 (2015). 814–815 , Parallel evolution of senescence in annual fishes in fishes annual in senescence of evolution Parallel Large differences in aging phenotype between phenotype aging in differences Large , 64–72 (2007). 64–72 , 14 2016 Vol. 4. (American Killifish Association,(AmericanKillifish 2004). 4. Vol. 296 l I Age-dependent regulation of tumor-related microRNAstumor-related of Age-dependentregulation . Insights into sex chromosome evolution and aging and evolution chromosome sex into Insights NTERESTS , 857–866 (2015). 857–866 , The African turquoise killifish genome provides genome killifish turquoise African The provides insights into age-dependent changes of age-dependentchanges into insightsprovides . , 681–691 (2013). 681–691 , J. Exp. Zool. Exp. J. Proc. Biol. Sci. Biol. Proc. |

shows a typical teleost aging process aging teleost typical a shows fishes as a new model system in biology. in system model new a as fishes 7 natureprotocols

, 181–192 (2014). 181–192 , 14

The authors declare no competing financial , 185 (2013). 185 , Nothobranchius furzeri Nothobranchius

Nothobranchius furzeri Nothobranchius J. Evol. Biol. Evol. J. BMC Evol. Biol. Evol. BMC 1 , 531–538 (2011). 531–538 , Nothobranchius furzeri Nothobranchius

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