Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. elfihhv oyopi ieyls hr h eai dl euaaetems osiuu om The with form. conspicuous cryptic, most usually the are are stages) medusa is (asexual adult distribution stages pelagic and polyp the ecology benthic where their small lifecycles, result polymorphic a have as Jellyfish and difficult (Tibballs waters death understood vaso- marine or poorly in vomiting/sweating, failure presently detection cramps, heart makes acute muscle elusiveness and pain, and doom back impending death, lower of to feeling include lead (Tibballs hypertension, can that syndrome possible symptoms Irukandji prostration, and with constriction, Envenomations injury, mortalities 2015). serious with al., years, or et 50 Kingsford (Ponce past 2002; syndrome’ the (Seymour, ‘Irukandji in countries dition Indo-Pacific deaths other 70 from least al. at reported et for and Australia recognised in increasingly responsible being now been has and species rine Bolte dangerous Brett for detection of method jellyfish viable cubozoan a as eDNA of Validation od ihe .&Moe,21) aycbzasaergre sdneosadee life-threatening regions even subtropical and and tropical dangerous in as resources regarded coastal of Kings- are managers species; cubozoans (˜50 to Many diversity challenges (Kingsford species pose low 2014). that relatively Mooney, animals with & jellyfish venomous of J. class Michael a are ford, jellyfishes) (box Cubozoa The jellyfish Introduction: Irukandji, both PCR, detect and DNA, swimmers to environmental to cubozoan, tool envenomation effective Keywords: of an risk the is reduce eDNA to Consequently, means ecology. the polyps. cubozoan provides of medusae approach of sivickisi detection This knowledge C. the our cubozoans. when by enhance of substratum explained the polyps near be and of shown medusae only concentration also the can between was the relationship This amplification in poor eDNA Queensland, absent. Positive a Island, were was jellyfish. Magnetic detection there of surrounding Positive waters surveys abundance removal. eDNA the visual and post-jellyfish in an days on eDNA medusae 9 Based and sivickisi to C. (September-January). C. up cubozoans and for spring/summer that xaymacana water four Austral demonstrated C. sea fleckeri, these experiments in C. detected degradation of for be DNA found each still Laboratory was could for but trials. quickly developed field degraded Carybdea were and DNA sivickisi, sivickisi Copula laboratory primers fleckeri, both (Chironex Species-specific species utilising offers was cubozoan validated study therefore four this approach barnesi). and for of method objective species Carukia The detection rare viable and life-stages. a detect polyp as xaymacana cryptic to eDNA of variation of potential presence use temporal but the the and medusae, validate has spatial pelagic to high waters (eDNA) only to not marine DNA due cubozoans, tropical Environmental difficult detect in to is presence abundance. potential cubozoans seasonal and of their detection occurrence The and their humans communities. in to coastal dangerous to risk are significant jellyfish a cubozoan poses of species certain from Stings Abstract 2020 18, June 2 1 ae okUiest olg fSineadEngineering and University Science Cook of James College University Cook James 02.Frhroe neoainfo nte 0cbza pce a euti h eiiaigcon- debilitating the in result can species cubozoan 10 another from envenomation Furthermore, 2012). tal. et tal. et 1 ui Goldsbury Julie , 02.Tog hs elfihsps ao elhrsst uas hi pta rareness spatial their humans, to risks health major pose jellyfishes these Though 2012). 08.Oespecies, One 2018). 1 enJerry Dean , hrnxfleckeri, Chironex tal. et 02 igfr ony2014) Mooney & Kingsford 2012; 1 n ihe Kingsford Michael and , 1 scniee n ftewrdsms eoosma- venomous most world’s the of one considered is hrnxfleckeri Chironex 2 h nycubozoan only the Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. ignDes lo n iseKt(el,TeNtelns.A54b rgeto h mitochondrial the of (Bayha Forward-16SL, fragment primers”, bp jellyfish a “universal 584 Magnetic the using A using of extracted Netherlands). PCR coast Reverse-Aa was The via 2010) the (Venlo, amplified (gDNA) was Kit off DNA gene Tissue either rRNA Genomic and 16S collected materials). Blood been (Supplementary DNeasy having Queensland Qiagen species Cairns, other or with Island, 2018), November (1 Queensland of Specimens Design Primer Methods: organism. oyscnb eetdwt DAa ie hnmds r betusing eDNA absent of are cubozoan amount medusa cryptic the when of if times presence ascertain if at detect and determine eDNA to field (4) with primers surveys; the detected eDNA; visual species-specific in be cubozoan from develop medusae can of estimated (1) cubozoan polyps rate as degradation detect abundance follows: the could with as determine eDNA correlated experimentally were if (2) aims determine cubozoans; specific these (3) of Our each of humans. presence to harmless is n lge nGniu Boatr,Acln,NwZaad n u noanBatsac downloaded search n-Blast a into put and Genome Zealand) Australian New the edited Auckland, to then (Biomatters, were sent sequences Geneious then Returned in sequenced. was Sanger aligned product be and to PCR Australia) Purified Brisbane, (AGRF, respectively. Facility Research primers reverse and forward namely, Australia; C. to tropical of used carybdeids coast the be east by could the that xaymacana along primers Carybdea waters PCR sivickisi, chirodropid marine species-specific in Copula validate occur fleckeri, and that Chironex develop species to focus cubozoan was the polyps. four study and detect benthic this cubozoans the venomous of not of objective medusa, occurrence The and detect ephyrae to ( approach on nettle eDNA been an sea only of Japanese has use the the on of works published medusae (Gaynor of of while detect presence (Minamoto life-stages Kyoto, to detect example, Bay, used For to Mazizuru been approach in jellyfish. has eDNA scyphozoan technique an including This as used species, organism reproduction. distributed an and by sporadically environment cubozoans death and (Kingsford the of excretion, envenomation in (Jerde of ecology metabolism, shed risks DNA rare normal the the of develop of on detection minimise to part research the to a important is enhance resources which is greatly coastal technology, it DNA of to Environmental tourism, managers potential assist to ranges. the to threat their has and expand related (eDNA) they will the as DNA blooms and cubozoans WalravenEnvironmental jellyfish risk (van of that expand detection of predicted will the level improve jellyfish been in that deadly has changes technologies of It range potential change. geographic the climate accurate the Given to and The related occurrence polyps. in understand is of cubozoans better intensify jellyfish to sources some of and the humans potential of to and envenomation dispersal medusa of populations risk (Kingsford of (Kingsford the local movements ecology minimising connectivity for the their small critical of (Schlaefer of is even level knowledge cubozoans populations of that a high detection other is a suggested connectivity with have relatively has of exchange can of levels research areas that little up geographic Recent populations’ have small made ‘local stocks may ‘metapopulation’ 2014). considered within a Mooney Moreover, be to may & them. corresponds bays among as generally connectivity 2014). such little species Mooney with a & ‘stocks’ (Kingsford of autonomous cubozoans range studying biogeographic of challenges The many the to contributed has alone ( detected minute been are have polyps where species .fleckeri C. tal. et hyar quinquecirrha Chrysaora < .fleckeri C. m n vnteautmdsi sxa hss fsm aacnb eysal This small. very be can taxa some of phases) (sexual medusoid adult the even and mm) 2 H16S 01 Keskin 2011; xaymacana scniee ihyvnmu n edyt uas n itm fenvenomation of victims and humans, to deadly and venomous highly considered is tal. et 54H(ne cirae 03 n hroyln odtosrpre o the for reported conditions thermocycling and 2003) Schierwater & (Ender 15141H eecletdfo osso a (19.1333 Bay Horseshoe from collected were , 08.Aohrjsicto o nesadn ouainsrcueadthe and structure population understanding for justification Another 2018). and tal. et .barnesi C. nBrea a,NwJre.Hwvr odt hr a enno been has there date to However, Jersey. New Bay, Barnegat in 06 Simpfendorfer 2016; tal. et nsitu in Hrwc 1991) (Hartwick fe xii yposo rknj syndrome; Irukandji of symptoms exhibit often 07 okda h plcblt feN odtc early detect to eDNA of applicability the at looked 2017) 2 and tal. et aui barnesi. Carukia tal. et . hsi eas eti uoonpolyps cubozoan benthic because is This 06,ivsv (Robson invasive 2016), 00;btciia ounderstanding to critical but 2020); ° ,146.8500 S, ouasivickisi Copula fteseisslce the selected species the Of ° ) antcIsland, Magnetic E), hyar pacifica Chrysaora stemodel the as tal. et tal. et tal. et tal. et C . sivickisi 2018). 2016). tal et 2017) 2016) ) . Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. i t95 10 at of min volume 5 reaction final a for itrpdladsac - m 6 a lcdi 0Lbce n=1)fildwt fartificial of L 7 with filled 16) = (n bucket L jellyfish 10 adult a single in a placed collected, was Once 16) (24/09/2019). = Queensland n North cm; Island, 2-3 trial, Magnetic this distance of In (inter-pedalia cubozoans. coast for (Thomsen the days determinations off 10 such to no hr 48 been from have anywhere to take Decay to removed. studies is using some jellyfish conducted the was once experiment degrade degradation DNA PCR An Step One Degradation Zymo DNA the 1: using Experiment purified USA). was California, DNA (Irvine, infor- process, Kit Supporting PPLPP Precipitate, Removal S3, the (Preserve, Inhibitor (Appendix through process (unpublished) workflow precipitated Burrows, cross a Once and using possible mation). Edmunds samples (PPLPP)), for water Purify detect sea Precipitate, to contaminants. from Lyse, sites extracted of the before was risk of filters DNA each the and Environmental pump at eliminate water sampling to the before (x3) equipment. through taken of passed water were was contamination reagent-grade solution, water with Longmire’s sterilised studies into rinsed of previous placed mL then in (Robson 500 and used where species hr invasive volumes controls, and Equipment 2 on rare for based of bleach bottles detection 10% plastic the L on 2 studies al. other sterile et and in lab collected our was in seawater field, the In replicate preservation technical single eDNA a and the detection. of sampling confirm positive amplification Water to a species, Australia) as all Brisbane, replicate For (AGRF, sample product. sequencing the PCR Sanger deemed for resulting sent the were of optimised product species-specificity at PCR ran 50 a 95 and at produced at probe that s min hydrolysis 15 the 2 of to of Curve compared Melt cycles when 50 specificity lower for the thermocycling to due chosen 1.4 was of volume consisting 0.9 reaction primer, each Forward with assay, Up ( Power species other all For Real-Time 5.0 5 of QuantStudio consisted a and on 0.9 Scientific) run ward), Fisher was (Thermo reaction machine qPCR PCR Each 1). (Table assay AACTATTCGCCTCAC-3’) For deem completed. To was reactions. species qPCR target PCR for the Supporting Quantitative repeated of S2, were (Appendix amplification species species exclusive each cubozoan a primers, other of on of against visualised replicates specificity min were technical 40 Amplicons triplicate surrounding for performed. V The waters 80 were information). from at species gel taxa each electrophoresis for agarose cubozoan replicates 1.5% other technical PCR, all Real-Time of Using DNA species-specific genomic of the specificity Queensland. The of against Island, gene. identification rRNA Magnetic assessed and 16s then mtDNA sequences the of was within alignment species primers on each for based sequences species Zealand) primer among New unique for Auckland, sites information). (Biomatters, designed polymorphic Supporting Geneious were S1, likely using primers (Appendix identify primers to eDNA eDNA and Species-specific species-specific taxonomy of their design confirm the to for suitable website NCBI the from .sivickisi C. 06 Uthicke 2016; μ ° Copula M ,3 t95 at s 30 C, h TPRaayi a ae naTqa yrlsspoe(Cop probe hydrolysis TaqMan a on based was analysis RT-PCR the , tal. et μ .flcei .xaymacana C. fleckeri, C. ees rmr 5 primer, Reverse M 16S ° ,1mna 60 at min 1 C, ° 08.Fralfil xeiet,pirt apig l qimn a okdin soaked was equipment all sampling, to prior experiments, field all For 2018). Rvre,0.25 (Reverse), R ,1 t60 at s 15 C, μ .Rslswr banduigotmsdtemccigfr4 ylsfor cycles 40 for thermocycling optimised using obtained were Results L. ° ° ,1 t95 at s 15 C, C. ° μ ,2mna 95 at min 2 C, apefrafia ecinvlm f20 of volume reaction final a for sample L μ Copula M < and %o h rgnleN ocnrto a enfudin found been has concentration eDNA original the of 1% .flcei .sivickisi C. fleckeri, C. 3 .barnesi C. μ ° .Ec apewsrni rpiae ae samples Water triplicate. in run was sample Each C. ilQ 10 MilliQ, L .sivickisi C. 16S ° 5sa 95 at s 15 , C tal. et rb,2.5 probe, P μ .sivickisi C. xTqah 0.9 TaqPath, 1x L ,eN eeto a i YRGreen SYBR a via was detection eDNA ), 02 Pilliod 2012; μ odtriehwqikyeN may eDNA quickly how determine to xPwrpSB re,0.9 Green, SYBR PowerUp 2x L , eua eecletda night at collected were medusae μ apead0.7 and sample L ° .xaymacana C. ,1mna 65 at min 1 C, tal. et tal. et μ Copula M 04;hwvr there however, 2014); 06 Simpfendorfer 2016; μ .Ti reaction This L. and siv ° ,uiiiga utilising C, μ 16S 16S fMilliQ of L .barnesi C. (For- F 5’- - P μ M Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. odciiy(S) eprtr n et CD,maueet eetkna ahst uigtejellyfish the during site Accordingly, each eDNA. at of taken detection were of measurements 4 level (CTD), influence depth at would and stored column temperature water and (PSU), the conductivity solution of stratification Longmire that of Individual possible was were mL replicates. It holders 1 and Filter in sites in placed collection. among of taken were contamination 5 h was filters of 12 Water with nylon chances within loaded (Sept-Nov). the filtered were season minimize then holders were medusa to filter which the replicate ice during each on were for placed changed they and containers as L h 2 (1830-1930 sterile dark after taken -19.17.102 also 19.15.332 3, 1, (Site (Site Bay Bay Nelly this Geoffrey to at close spots’ stay 6 generally hot and (Garc´ıa-Rodriguez – spring copulation 3 alga Austral 1L following was macro the released into column in the habitat collected water by this (Schlaefer covered were total over habitat largely abundant the samples preferred reef are substratum; water medusae coral the stratified adult over above as Depth targeted matrix, just by was analysis 2019). and separated Sampling eDNA (June surface sites deep. for winter the three Water m near Austral at absent. Niskin the Queensland, were Oceanics during Island, determine adults General m to Magnetic when 400 model surrounding polyps - waters good cubozoan 300 a nearshore of provided from presence therefore, collected the species, was detect This could period. eDNA spring-summer if the to restricted 2020), of presence stages The life of Detection 3: through Experiment pumped were (Renshaw 4 they solution at preservative where of stored Longmire’s radius lab were into m the placed 2 to and a back within forceps taken and and surface ice the and of on adult m 20 stored containing 0.5 a were within for either Samples water evidence light. collecting historical containers JCAM (Schlaefer L to the (September-January) 2 due spring/summer sterilised sampling using Austral for taken the chosen data in were during historical bay boat Island, medusae to the (creek, in due Magnetic present chosen meters Bays, commonly was were Florence Bay of For medusae (Dec-Feb). Horseshoe hundreds that months bay). indicating For several summer the (SLSQ) sampling: the of by Queensland Water side Saving b.) separated northeast Life minutes. and Surf sites 2 from net m of four stinger 3 course the from of a inside attract over Queensland, depth ramp, to light a Island, min the 30 within of Magnetic for radius abundance to Bay, left m jellyfish attached and 2 of water lumens) a of estimate within (2000 fleckeri, m an and 2-5 torch obtained surface in LED the sites snorkeler an the from A of had sampling. each ‘JellyCam’ for at lowered Each cubomedusae were jellyfishes. devices The of crates. abundance weighted and presence the eeatatdt ihsuig‘elCm’(Schlaefer ‘JellyCams’ using lights to attracted were bnac siae:Oe h oreo ihs eua eecletdi nw elfih‘hotspots’ Queensland North jellyfish Island, Magnetic known near waters in shallow in collected metres were of hundreds medusae (19.1359 to tens nights, by separated 5 were Sites of course the (Schlaefer Over estimates: tubes Abundance the mL determine experiment 2 to Field sterilised jellyfish 2: in of Experiment removal placed water the were (Renshaw of post analysis. papers solution filtering 9 and samples Filter The and Longmire extraction all column. filters. 6 of future that nylon water 3, mL um 0, so the day 1 5 once in on through containing sampled remaining undertaken filtered was eDNA was being 4) time) of water = per concentration of (n buckets L bucket 4 7 Each = full removed. (n the was with medusa independent the were h 12 After saltwater. .barnesi C. o ae ape eetkndrn algthusi h utainsme Jnay nHorseshoe in (January) summer Australian the in hours daylight during taken were samples water ,146.842 S, tal. et μ ( m .sivickisi C. yo le ihn1 fcleto.Oc lee,fitr eermvduigsterilised using removed were filters filtered, Once collection. of h 12 within filter nylon ) o .fleckeri C. 00 ewe etme nNvme for andNovember September between 2020) C. o )(al ) h eua of medusae The 3). (Table E) tal. et ape u ohg ubdt fsml) r5 or sample), of turbidity high to due samples euai aessronigMgei sadi ihysaoa (Schlaefer seasonal highly is Island Magnetic surrounding waters in medusa o ,146.84.831 S, μ 00.Acrigy hshbtti sweeeby ude ol ieybe likely would bundles embryo where is it habitat this Accordingly, 2020). yo les fe lrto fec apeadeupetbak,the blanks, equipment and sample each of filtration After filters. nylon m .svcii .xaymacana C. sivickisi, C. o o ) antcIln.Smlscletddrn h itrwere winter the during collected Samples Island. Magnetic E), 146.86479 & S tal. et tal. et 4 .sivickisi C. 08.Wtrsmls( )wr ae rm‘jellyfish from taken were L) (2 samples Water 2018). tal. et 05 Williams 2015; 00;i hswyi a osbet estimate to possible was it way this in 2020); o .sivickisi C. and ;St 19.15.486 - 2 Site E; and tal. et .barnesi C. .xaymacana C. o o N extraction. DNA for C tal. et 05.Alevrnetlsamples environmental All 2015). n n ih for night one and tal. et μ ( m 06 n trda 4 at stored and 2016) el,Gory rhrand Arthur Geoffrey, Nelly, , 00.Wtrsmlswere samples Water 2020). .svcii .xaymacana C. sivickisi, C. Sargassum o r htpstv and photopositive are ,146.86.183 S, .xaymacana. C. n edcoral dead and o )and E) o for C tal. et C. Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. ea neN,orfidnsidctdafs erdto fjlys N ihntefis aso the of days 3 replicates. some first in the days 9 within after DNA left microbial jellyfish signature on species residual of focused a a only degradation only if which with fast identify trial, treatment, a laboratory the help from indicated simple can removed a findings environment being on our animal aquatic Based eDNA, the time. certain on in to a decay degrades how within DNA therefore present and which to been medusae at has dangerous for rate are areas risks. the source cubozoans envenomation the Understanding jellyfish many polyps on manage as knowledge cubozoan potentially of significant, detect better paucity is to and a of finding eDNA is monitor time there particular for the currently This potential and at the populations. humans locations highlight medusae proximate to adult in first to jellyfish the of also presence on is eDNA known based an detection study or using confirmed The systems observations analyses marine sampling. visual eDNA in water and against jellyfish cubozoans cubozoan field four small the of for in detection designed the were on primers report Species-specific to approach. first the eDNA is study the presence This constrained the thermocline by shallow explained a be that only likely Discussion: can 3a). is signature (Figure It surface eDNA substratum. the the coral of below sampling, the m signature of 0.5 within time within hidden the collected polyps at samples of present water not in are made only of species was eDNA detection The Positive substratum. detected. samples. the being water in halocline significant detected of was no medusae with adult m the 2 also When at was detected species was 2). this thermocline (Table said, reverse eDNA A being Surf with That from detection abundance. tows zero approach. with species in eDNA concurred low collection an this with and using and observations sites detected to two due still at area only the between however, in surveys; correlation be no to Queensland. was known Lifesaving There were they 11). (‘blind’ although 10, jellyfish visually, attract (sites to detected used xaymacana were still C. lights no was sites sites some At detected. these 4. was of from and eDNA abundance 3 their eDNA 1, occur, sites but to at known samples), replicates were of jellyfish 100% of in species detected four the where general, In each field: of level the subsampling in the at Detection Variation containers variation. ( between the small of differences was 65.7% significant bucket explained were replicate this There and days (48.2 days. within levels among (replicates) initial difference were of significant levels % indicated eDNA 12 ANOVA 9, about Day to By 3 Day 1). by decay rapidly to observed. was (0.08 cross-amplification DNA North No Townsville, surrounding species. tested waters target was the the experiment set in from Degradation primer found product Each PCR species amplified cubozoan cubozoan. only other and target all Queensland, each against for PCR developed exclusion using successfully were primers Species-specific water column. the water the in Primers through haloclines DNA and of thermoclines dispersal of vertical Results: presence/absence the the inhibit identify potentially to could used which were column, data These season. .sivickisi C. ± .sivickisi C. 53 go eDNA/ of ng 55.30 and .fleckeri C. samdl hspoiigagntcto olct oreresta rvd recruits provide that reefs source locate to tool genetic a providing thus model, a as aydaxaymacana Carybdea euai h edadqatt fDA(iue2 .4 f=2;P 23; = df 0.04, - = r 2, (Figure DNA of quantity and field the in medusa < .sivickisi C. 1%). .sivickisi C. eerltvl aea h ieo apigand sampling of time the at rare relatively were μ < )wsdtce nbcesfo a frmvlo elfihadobserved and jellyfish of removal of 0 Day from buckets in detected was L) %o nta ocnrtosadi oebcesudtcal.Anested A undetectable. buckets some in and concentrations initial of 1% eentpeet(..i itr,a DAsgaueo hsspecies this of signature eDNA an winter), in (i.e. present not were aui barnesi Carukia a o eetda h ufc Fgr ) ie eua fthis of medusae Given 3). (Figure surface the at detected not was a eetdb ohJAS swl swt snorkelling with as well as JCAMS, both by detected was 5 a ee iulydtce uigsmln and sampling during detected visually never was ± 39n/L hr hypaeud(Figure plateaued they where ng/uL) 13.9 .fleckeri C. a o detected not was .sivickisi C. > 0.05). was Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. ntesme esn hsaiiyt o eetallf itr tgswl rvd e potnte to opportunities new provide will medusae stages adult for history sources life as all serve detect that now reefs detect to polyp to ability of first This identification the the is season. for study summer allows Our turn the through visibility. in in water particles This low DNA located. and of polyps be habitats movement of complex presence the DNA passage likely size, inhibit of vertical the beds small may amounts the polyp nature, blocking small therefore of cryptic for preventing and Location their known or 2003) well column. the reducing Kingsford are water of barrier & thermoclines m the a 0.5 as (Gray as benthic within surface, particles taken the acted the Critically, of samples reaching thermocline including species. substratum of the that the the eDNA likely sampling of from stratified any is polyps depth Accordingly, It of in presence detected time. substratum. proximate be the the only during by at embryos could present explained of planulae polyps were be bundle release only medusae a they could adult drop assume detected females polyps, we adults, for when and of sampled winter, (Garm mating 2020) we during substratum the substratum sites Following the the the (Sept-Nov). to on of season close of all taken jellyfish presence At samples previous proximate water the absent. the in were signature detect medusae eDNA to an adult utility a detect have provided to to site able shown a were was inhabit approach to eDNA eDNA known using An example, were revealed For were jellyfish detection. detections useful, lights. where of positive in value Samples are but positive detected combined detection. counts, true be techniques visual visual to two and without likely the imagery even less JCAMS techniques, but provided are in eDNA, JCAMs rare and the than relatively rare Clearly was abundance are abundance. relative jellyfish as of when well particularly estimate as accurate jellyfish, of more presence/absence a the determining for sites. useful within (Schlaefer replicates (JCAMS) water and JellyCams sea location stratification (7) within vertical sites thermocline; locations, (6) the (Furlan among through eDNA dispersion; levels, (Gargan (Lacoursi`ere-Roussel of close transported and temperature nature source observed being with concentration increase clumped the were from may The in from source jellyfish eDNA the eDNA role from as the detectability of preventing study as a dilution temperature, column current have of water the may speed the in oceanography the of case microbial (4) scale the for taken; small time be were more (5) samples not leaves this the may organisms, where this source away; to though from distance distance decay; some some physical or taken and nearby been in have organisms DNA (Furlan samples from of were where eDNA that distribution (3) we of be heterogenous where known, nature the not species clumping sampling. (1) is the those following: before samples the to of field in to due abundance the due column and in probably water DNA present countswas the laboratory of visual was quantity the obtain species between to both target able relationship in the poor DNA knew The jellyfish we observed. of taxa detection jellyfish sivickisi the most from Copula For in advected act field. successful be currents forces the was can effect oceanic and approach eDNA true that that DNA the distance and environmental determine the days An To determine 3 DNA. to eliminate first required and the is source. disperse in modelling a rapidly dispersal, decline field and eDNA fastest the jellyfishes on locations. in with have of proximate Minamoto detect rapid, from eDNA to from currents is the sought by findings jellyfish on we carried the in been organisms with has degradation target consistent eDNA the DNA These and/or is recently, DNA detected. data area is of the our remain eDNA decay in Furthermore, if can rapid been organism DNA the had an or to of that (Thomsen were, proximity due factors concluded close that aquatic confirming been suggest and further has findings DNA dilution is of on degradation it depending the where days, affect 10 taxa to other 2 on Pilliod for studies environments to aquatic similar in were findings Our tal. et 04.I h cai niomn,cret,U aito,dlto n irba ea all decay microbial and dilution radiation, UV currents, environment, oceanic the In 2014). , .xaymacana C. tal. et nsitu in tal. et and 05 sterhm agsaei h eso ers(Schlaefer metres of tens the in are ranges home their as 2015) n hssoseN rvdsasrn rdco o hr oyswill polyps where for predictor strong a provides eDNA shows thus and .fleckeri C. tal et 00 n onso elfihwti e erso h ihswere lights the of metres few a within jellyfish of counts and 2020) nsitu in 06 loepaie h motneo elcto tmultiple at replication of importance the emphasises also 2016) eedtce tmlil ie hr hyhdbe visually been had they where sites multiple at detected were o otcbzashseue cetsst ae u to due date, to scientists eluded has cubozoans most for 6 tal. et 06;()tesuc()o l eDNA all of source(s) the (2) 2016); tal. et 21) ugsigtert of rate the suggesting (2017), .sivickisi C. .sivickisi C. .xaymacana C. tal. et oys We polyps. tal. et tal. et htwas that 2016). 2017); tal. et 2012; Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. f9dy.Fleigwsudrae ,3 ,9dy otrmvlo elfiht eemn h aeo DNA of rate the determine to day. each jellyfish (ng/ at of DNA eDNA removal of of concentration post level days for 9 bars 2: 6, Table as 3, shown 0, are errors undertaken Standard was Filtering degradation. days. 9 of 1: Figure Type Assay (bp) Fragment PCR Length barnesi C. Sequence xaymacana C. fleckeri C. Primer sivickisi C. TaqMan). or (SYBR type assay Species the as well volunteers. as from fragment, field PCR the final in of assistance (bp) the protocols for sharing grateful for 1: Budd Table also Alyssa are and We Cooper Madie project. Rath, Villacorta to the Cecilia throughout utility thank to high like and would of authors cubozoans change. The be climate adult also of seeding result will locations a approach of source as This Acknowledgments: eDNA cubozoans spatial the of ecology. detect detect broad shifts jellyfish to could a range of used have we potential understanding detect that be that our can jellyfish was fast-track therefore detect finding potentially DNA to major Environmental way A labour-intensive . to less variation. technology Environmental effective and temporal life-stage. an polyp cost-effective is and cryptic approach the a eDNA putatively and provides an medusae that DNA both demonstrated systems, was marine is in cubozoans study detect current the in cubozoans. conclusion, of In use habitat and ecology the understanding pce-pcfi tN 6 RApie eune o orcbza pce,soigtelength the showing species, cubozoan four for sequences primer rRNA 16S mtDNA Species-specific il aiainicuigvsa onso uoonjlys pce tec oainwt average with location each at species jellyfish cubozoan of counts visual including validation Field uniyo N (ng/ DNA of Quantity Car Chi Cop Cop Car Car Car Chi Cop flec flec bar bar xay xay siv siv siv 16S 16S 16S 16S 16S 16S 16S 16S 16S μ )fo lee ape.Alfil elctswr olce rmmrn areas marine from collected were replicates field All samples. L 2 filtered from L) ’CGCACTATGAC3 TaqMan 199 5’-CACAATTATGGGGCGGAA-3’ 5’-CTGTCGAGCTTAATTGGTATC-3’ R 5’-AACTATTCGCCTCAC-3’ F P ’GCGTACAACT-’10SYBR 180 5’-CGATTCAATATCACTGGGAGGCAAG-3’ 5’-GACTGTTTACCAAAAACATA-3’ R F ’TAGCGTATAT-’20SYBR 280 5’-CTTCTTTCCCCGATCAAACCAAC-3’ 5’-TGAGGCCTGCTCACTGATTC-3’ R F ’TTTTTGACAGTC3 2 SYBR 127 5’-GACCCACAGATTTCGTGACTG-3’ 5’-TCTATCTGTTGCAACAAAGGTCC-3’ R F μ )atrfitrn fwtrfo oridpnettnsoe course a over tanks independent four from water of L 7 filtering after L) 7 .sivickisi C. polyps nsitu in Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. aui ans 0/01)St 0 0 0 3 0 NA 0 0.01 3 2 4 Site (07/10/19) 0 2 2 6 Site barnesi Carukia (07/10/19) 2 0 Bay of side Northeast xaymacana Carybdea (17/01/19) 100 Replicates eDNA Positive # Replicates eDNA Field No. jellyfish of 1 Count Site fleckeri Visual Chironex (1/11/18) Name Site Blind date Collection Location sivickisi Copula for eDNA comparing when attempted Species were counts visual no excluding Note: sites, All JellyCams. utilised Queensland. sites North Island, Magnetic surrounding rhrBay Arthur Bay Florence Bay Geoffrey Bay Geoffrey Bay Horseshoe effe Bay Geoffrey Bay Arthur Bay Florence Bay Nelly Bay Geoffrey 2/91)St 1 0.02 0.02 0.04 0.04 2 2 1 3 2 1 3 3 3 3 2 2 15 32 115 21 150 125 7 Site 6 Site 5 Site 4 Site (07/10/19) 3 Site (07/10/19) 2 Site (24/09/19) (24/09/19) (15/11/18) (15/11/18) 1/11)St 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 3 0 3 1 3 3 0 NA NA 0 NA 0.02 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 9 Site 2 3 0 8 Site 0 3 2 7 Site 3 1 6 Site 2 (14/11/19) 5 Site 3 2 (14/11/19) 3 (14/11/19) 3 (14/11/19) 3 (07/10/19) 0 2 0 0 0 2 2 0 2 3 0 2 7 0 Site 0 3 5 0 Site 4 0 Site (07/10/19) 3 0 Site 2 Site (07/10/19) 1 Site (24/09/19) 5 Site (24/09/19) 4 Site 0 0 (24/09/19) 3 0 Site 0 (24/09/19) 2 Site 0 (07/10/19) 1 Site (24/09/19) 15 (24/09/19) Ramp Boat (24/09/19) Net Stinger (24/09/19) Creek 11 Site (17/01/19) 10 Site (17/01/19) (17/01/19) 8 Site (07/10/19) (15/11/18) (07/10/19) 0/01)St 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 3 3 3 3 2 3 3 3 3 0 0 0 0 0 1 0 1 3 1 Site 2 1 Site 1 Site 13 Site (07/10/19) 12 Site (24/09/19) 11 Site (24/09/19) 10 Site (24/09/19) 9 Site (24/09/19) 8 Site (14/11/19) (14/11/19) 3 (14/11/19) (14/11/19) 630 9 Site (17/10/19) 8 hrnxfleckeri Chironex ape n ‘blind’ and samples .fleckeri C. . < < < < < *1000 (νγ/μΛ) ὃνςεντρατιον ΔΝΑ Αvεραγε < < < 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. ieetsymbol. different a (ng/ 2: Figure μ )a h epciest ihnGoryadNlyBy antcIln.Sts11 r ersne by represented are 1-11 Sites Island. Magnetic Bay, Nelly and Geoffrey within site respective the at L) orlto ewe iulaudnecut of counts abundance visual between Correlation 9 ouasivickisi Copula n vrg uniyo DNA of quantity average and Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. oi ffcso hyar unucrh Ciai:Syhza and Scyphozoa) assays. the of (Cnidaria: cell-based study using quinquecirrha Comparative venoms Chrysaora (2015). JJ of Dorantes-Aranda effects CE, stream- Wright toxic K, a Luna-Ramirez from DL, eDNA Brinkman of D, detection Ponce influencing Factors (2014). LP Waits, amphibian. RS, dwelling Arkle CS, Goldberg DS, Pilliod (2015). D. Jerry, R, tool Saunders S, H, Yamamoto, distribution. Robson S, T, jellyfish Hidaka, Noble temporal A., and Fujiwara, spatial R., reflects K. DNA Katsuhara, from ronmental M., biomass Fukuda, and T., abundance Minamoto, fish conditions. environmental Estimating and methods (2016). the capture Resources, L near among variability and Bernatchez, concentrations: on M, eDNA cubozoans (Vol. Rosabal, of A, Blooms patterns Lacoursiere-Roussel Jellyfish Abundance In (2012). (Cubozoa), MD Reef. Barrier O’Callaghan, jellyfishes Great JE, box Seymour of MJ, ecology Kingsford Stake- The Empowering (2014). (2018). CJ AA 9789400770157). Mooney, Yangihara eDNA KA, MJ, Perspective. using Pitt Kingsford A species VL, Jellyfish: fish Fuentes Stinging freshwater C, Manage invasive Bordehore to and holders S, rare Becken of MJ, Detection Kingsford revised. (2016). ichthyofauna HH Iznik Atar Lake species box-jellyfish EM, pyrosequencing: aquatic rare the Unal species. of aquatic E, of detection rare Keskin “Sight-unseen” stages of (2011). surveillance life DM eDNA Lodge early DNA: WL, environmental the Chadderton using AR, of Mahon behaviour CL, Jerde and ecology Distributional fleckeri Chironex (1991). stage RF history life Hartwick, devil early of of detection qPCR study (2017). case CL a Barry quinquecirrha DJ, PCR: Restaino PAX, quantitative Bologna JJ, and sensitive Gaynor a eDNA of Development using biodiversity (2017). seamounts. J pelagic at Carlsson survey ray JEL, Carlsson to JA, method Finarelli CK, detection Pham T, Morato jellyfish LM, species Gargan box fertilizing of internal histology between Gonadal variation sivickisi (2018). edna Copula reveals AC of Marques Cubozoa) sensitivity JEAR, the Marian (Cnidaria: estimating C, for Ames framework Lewis A Garc´ıa-Rodriguez J, (2016). RP Duncan CM, morphology. Hardy molecular surveys. D, from Gleeson evidence EM, cnidarians: Furlan derived not evolution, are and and Placozoa 18S biology (2003). Molecular of B Analyses Schierwater Phylogenetic A, Ender and Sampling Taxon Among DNA. Complete Relationships (PSU) Ribosomal Evolutionary on salinity 28S (2010). Based HDH (b) Families Steven MS, and Jellyfish Barbeitos Scyphozoan sampling. AG, temperature Collins of MN, (a) time Dawson KM, with the Bayha at along column Island, water the Magnetic of surrounding profiles waters (Deep) benthic 3: Figure naieaia R rjc,JmsCo nvriy onvle Australia. Townsville, University, Cook James Project, CRC animal Invasive . oeua clg Resources, Ecology Molecular 16 Lf)Qatt of Quantity (Left) santls nBrea a,NwJersey. New Bay, Barnegat in nettles) (sea 6,1401-1414. (6), (Tripedaliidae). . Hydrobiologia, aieBiology, Marine Hydrobiologia, oeua clg Resources, Ecology Molecular nertv n oprtv Biology, Comparative and Integrative ouasivickisi Copula Toxicon, 20 ora fMorphology, of Journal 216-217 690 164 1,130-134. (1), 16 1,257-268. (1), 5,1. (5), 106 3,641-654. (3), 1,181-188. (1), 57-67. , DA(ng/ eDNA ia eot h tlt feN satlpasurveillance tilapia a as eDNA of utility The Report: Final iceia ytmtc n Ecology, and Systematics Biochemical 14 10 osa Management, Coastal 1,109-116. (1), 279 ora fCatlResearch, Coastal of Journal 50 μ 6,841-856. (6), )we ape rmsrae(hlo)or (Shallow) surface from sampled when L) 3,436-455. (3), lsOne, Plos hrnxfleckeri Chironex osrainLetters, Conservation 46 ltn alata Alatina 12 1,1-18. (1), 2,15. (2), Ciai:Cubozoa) (Cnidaria: 78 tal. et 67 oeua Ecology Molecular 7) 184-192. (78), Aaiia)and (Alatinidae) 29-36. , 4 21) Envi- (2017). 2,150-157. (2), chrysaora Posted on Authorea 18 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159248732.24076157 — This a preprint and has not been peer reviewed. Data may be preliminary. ilasK,Hyar P igi J(06.N les ofigs ehdfrpeevto fwater of preservation for method A fridges: no filters, No analysis. (2016). eDNA AJ for Piaggio samples KP, Huyvaert KE, area. JWP, Williams Sea Coolen North of PC, differentiation southern Luttikhuizen population the and A, in distribution Bol identification, polyps Solaris) J, Molecular Bleijswijk cf. polyps? van (Acanthaster the F, are PCR. seastar droplet Driessen corallivorous digital L, of Walraven using detection van reef eDNA causing barrier jellyfish great (2018). carybdeid the JR on Australian Doyle outbreaks M, (2012). Lamare D. S, K. Uthicke Winkel, LA, diverse Gershwin a R, of syndrome”. Li Detection “Irukandji HA, (2012). samples. Tibballs E seawater J, Willerslev from Tibballs M, DNA Rasmussen environmental PR, using Moller fauna LL, fish Iversen marine J, Kielgast wild. PF, the Thomsen in sawfish R., Lindsay largetooth RK, endangered Basiita critically J, detects Goldsbury localised TH, dna Noble highly PM, Kyne of venom CA, Simpfendorfer maintenance of touch Behavioural One ecosys- (2002). freshwater (2020). J tropical MJ Seymour for Kingsford in Fine-tuning S, detection Yadav (2016). ( fish E, jellyfish DR invasive Wolanski Jerry for JA, DW, technology Schlaefer Burrows eDNA SKA, of Robson Application RJ, tems. DNA Saunders tropics: alcohol TH, the phenol–chloroform–isoamyl preservation Noble temperature a HLA, room into Robson The assimilation (2015). and DM samples Lodge, DNA extraction. MM, environmental McVeigh CL, filtered Jerde of BP, Olds MA, Renshaw oeua clg Resources, Ecology Molecular ouasivickisi Copula oeua clg Resources, Ecology Molecular Toxicon, ls uoo)populations. Cubozoa) class , M eerhNotes, Research BMC 59 6,617-625. (6), 16 4,922-932. (4), aieBiology, Marine , 15 111 1,168-176. (1), , 72-75. 9 11 1,298. (1), 163 aieBiology, Marine 172-172. , oa Reefs, Coral nagrdSeisResearch, Species Endangered lsOne, Plos 167 37 (4). tal et 4,1229-1239. (4), 7 8,e41732. (8), 21) Environmental (2016). . uei aurita Aurelia tal. et 21) Where (2016). 30 109-116. , jellyfish