Posted on Authorea 10 Feb 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158137013.34385109 | This a preprint and has not been peer reviewed. Data may be preliminary. ilgclfcos(.. rdtr,prsts n opttr)drn h avlsae Sih&Wootton, & (Smith stages larval larval the influence during and temperatures) that competitors) extreme factors and and contrast, rates In important parasites, oxygen 1995). 2010). low predators, also Janzen, (e.g., (e.g., are & abiotic Refsnider factors to care choice 2005; vulnerable biological parental Kitamura, the are the 2002; with care individual, Reynolds, parental oxygenating an species & without or of Mills species in eggs 2000; success spawning al., their reproductive et of defending the (Smith by survival site Besides instance, and 1995). for sites Wootton, type larvae, spawning & of of prepare (Smith and rate them select survival around to and water strategies number Davies, reproductive the different 1998; increase use Robinson, to care parental & with Rothstein (e.g., fishes 1998; Oviparous interactions Takasu, host-parasite 1992; on Burdon, studies of from & adaptation evidence mainly 2000). (Thompson Theoretical the obtained parasitism) 2006). in been al., brood role have et symbiont avian Liu important traits and 2002; adaptive host-parasite, an (Thompson, Prey-predator, and plays coevolution coevolution 1994). of and (Thompson, examples organisms typical populations interrelationship living are the associated relationships all from almost and resulting of organisms changes speciation evolutionary of and reciprocal group of a consists that between process a is Coevolution INTRODUCTION 1. larvae bitterling the that evidence after direct 4. days first mussel. 24 mussels. a the At the are from days of develop. ejection tubercles out 11 to premature minute swimming cavity began prevent the observed fin suprabranchial to that were caudal adapted mussels’ prove individuals and morphologically to presented some are rate migrate herein and heart to interlamellar results disappeared, of mussels’ experiment almost components began clear the had The and had tubercles in hatching, larvae minute remained after the the larvae until days time, hatching, stage) The this 10 (reduction At until reduction 3. day hatching. gradual demibranchs after final stage). 1 gill a develop (disappearance the through to hatching abruptly went of stage), began after and (peak space days days stage), tubercles 6–7 reduction 27 These after (abrupt height disappearing days are peak tubercles. completely the 8–10 tubercle minute reached after stage), minute the height (growth days of of in their 2–5 reduced development function larvae, for grew pseudosericeus the morphological stage), Rhodeus the and (formation of host hatching of mussels, after stages the evidence inside developmental leave direct the are migration they which as of before their larvae, analysis presented larvae characteristics, of comprehensive free-swimming physiological surface a in study, skin and disappear present the morphological and the on ability In tubercles swimming minute adaptations 2. weak morphological the have mussel. with are and larvae mussels adaptations host early-stage well-known in in most eggs developed place The to survival. ovipositors larval long use increase that to fish freshwater small are Bitterlings 1. Abstract 2020 5, May 1 mussels Kim host Hyeong by ejection an premature as prevent tubercles to minute adaptation larvae’s bitterling of evidence First ffiito o available not Affiliation 1 1 Posted on Authorea 10 Feb 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158137013.34385109 | This a preprint and has not been peer reviewed. Data may be preliminary. a opie fsl n u.Test a ih pce ffehae se,btol n bitterling study. one this only perform permission to but received 2017-22) we fishes, number, bed thus (Permission freshwater Korea, stream Korea of of of The Environment Environment the species stream. of of Heukcheon eight Ministry Ministry the the the had by from to site species connected endangered The an respectively) mud. considered wide m, m and 0.8 (30 silt and pond species, small m of a 1.5 comprised was Kyeongki- of site was Yangpyoeng-gun, experiment in depth river The mean Namhangang 2017. the and of March–April stream in Heukcheon Korea the do, at performed was study This site of development Study the survival.2.1. of better advantages METHODS the for evolutional mussel & of the MATERIALS inside height discussed larvae 2. the I of Further migration the among in and and mussels. relationships development, tubercles tubercles the the during the minute minute in larvae the on in of larvae the development Therefore, focused the of the characteristics of I physiological that conducted. position this, and indicating been For morphological evidence have tubercles, direct tubercles ejection. minute find minute premature to the prevents was of bitterlings goal development migration my the their study, characteristics, correlating and present physiological studies to and mussels, comprehensive assumed morphological no the were their However, tubercles with inside larvae mussels. minute of host the and stages their results tubercles developmental from their minute the larvae 1988a, the on of Jeon, of based ejection development & and premature the larvae, prevent (Suzuki described 2008). of projection briefly al., characteristics have wing-like morphological et insemination a & the Park vitro forming 2008; in Suzuki larvae, using al., studies of et 1985; the Many in Kim eyes al., developed 1990; the larval mainly et 1989, on the are 1988d, though and Suzuki tubercles 1988c, even minute from larvae 1988b, 1985; the bitterlings, larvae the that of the of 1984a, reported stages preventing have part developmental Hibiya, in studies frontal all Previous & ‘minute role in diverse. called common important (Suzuki is are cells, an morphology gills tubercles play epidermal Minute the to single-celled 1987). develop known from Jeon, larvae are ejected sophisticated fish which prematurely make The surface, Spence being Bitterlings skin 2018). 2006c; their 2006a, al., Kitamura, on 2013). et 2004; tubercles’, al., Methling Smith, et 2013; Smith & morphological 1997; Smith, and Spence (Aldridge, & behavioral, mussels physiological, host 2003; on unique spawning several Reynolds, interaction have for host-parasite and adaptations & ejection of prevent type Mills to a decisions 2001; fact oviposition in al., is al relationship et et bitterling-mussel the Reichard (Reichard that 2003; shown have Reynolds, studies & Recent between Mills of coevolution 1997; choice of example & al., and notable (Refsnider swimming et interaction a studied acquiring 2017). (Reynolds is sufficiently Host-parasite mussels after been parasite and exit not 2008). and bitterlings have only between vulnerable Kitamura, host that relationship larvae most ecology 2004; a The the vertebrate are al., when of 2010). where fish Janzen, et aspects and from the critical Smith mussels, spawning are which relationship 1982; site the in of oviposition Neves, host-parasite inside period moment & safely The the the (Zale spent however, ability during is eggs. occurs; eggs) mainly few mussels as by difficulties very (i.e., larvae have presents of they mussels ejection care, and premature parental fish have these 1999; become not (Aldridge, between they where, feeding do until mussels external reserves bitterlings begin of own Although and cavity their mussels gill on the through 2004). the leave feeding al., larvae siphons in weeks et the coloration inhalant fertilized 3–4 stage, Smith nuptial for mussels’ therefore, this have remain the at are, who larvae; they enters eggs free-swimming fish, temperature, The that male the sperm The on actions. release depending breathing siphons. mussels, and exhalant the feeding spawn mussels’ around and their the ovipositors territories 2004; their through elongate al., form mussels bitterlings et and female of (Smith the gills Unionidae) season, spawning (Bivalves: the the mussels on During freshwater Northeast with 2007). and relationship al., Europe et unique Damme in a have distributed they predominantly and fish freshwater Asia, small are (Acheilognathinae) Bitterlings hdu pseudosericeus Rhodeus n n uslspecies, mussel one and , 2 nodulsa sinuolatus douglasiae Unio . 00 oce tal et Rouchet 2010; , × . 0mln;maximum long; m 10 .pseudosericeus R. . is , Posted on Authorea 10 Feb 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158137013.34385109 | This a preprint and has not been peer reviewed. Data may be preliminary. hratrrfre oa H) 2 ufc fwn-iepoeto opsdo aro osladone and dorsal head of pair and a eyes covering of composed projection projection sac wing-like yolk three of into Anterior divided surface (1) was (2) (2008): larvae EHR), the al. as of et to surface Kim the referred vision following patterns, (hereafter Zeiss distributional 3) Carl the (Figure SEM. of a regions during Zeiss, description tubercles, used Carl the was minute (Supra40VP, facilitate Germany) SEM To the CO Zeiss, liquid under of Carl with examined 4.4, assessment point REL. then and critical (LE and a documentation camera gold carcodylate-buffered to SEM photographic under with dried the For h sputter-coated and For 24 Germany). were series, measured. for using graded samples was fixed ethanol determined dried were tubercles an development was The minute larval in stage of the dehydrated developmental stage glutaraldehyde, of each larval 2.5% height at each the specimens at and three tubercles analysis, (SEM), minute microscopy the electron scanning of trend development tubercles The minute larvae’s demibranch. the gill Kim of following Observation the Germany), 2.5. Zeiss, to Carl was cavity 4.5, (version position suprabranchial program (2006). larvae’s LE the al. the AxioVision the et from of other the Moreover, using length the length Japan) 1. transverse in longitudinal SMZ-10, the the (Nikon, parts and of the measuring three 2 stages divided and by into developmental by was Japan) divided mussel The followed gill (Figures also II, the the outlet, was parts Mark cavity, of it the (Canon, nine suprabranchial siphon (U); photographed from into the part and farthest divided with upper recorded contact were the and accurately of gills being (M), point part the 3 its middle mussels, direction, to (L), the demibranch part in gill lower into position the larval from with 2); in stage Mussels developmental 1, changes tanks. and cm. different the number, 1 in evaluate position, approximately housed the To to were for spawned opened examined the without were be of and Mussels to inner) larvae. cut for mussels or muscle of checked enabled adductor outer were that their right, days had or device 30 spawn (left mussel-opening for gills a day four using per the by mussels on three larvae tanks, of the presence in day 1 After temperature water keep of live to Observation a the used 2.4. with induction, were daily spawning heaters fed dissolved For that aquarium and so aquarium. aquarium and supplied the experimental was mg/L, Oxygen of 7 tanks. bottom 20 above glass three around the maintained in was on placed separately height) (DO) were oxygen cm mussels 50 (10 of width spread groups cm three evenly (60 was aquarium sand experimental and an laboratory, the In aquarium the in rearing pond Mussel box generator. the 2.3. plastic oxygen from the an Water later, length with experiment. hours cm each, laboratory 36 (100 induction mussels a 2017, boxes spawning 50 March to plastic the with 29 three boxes complete of in three to morning collected was into removed the was experiment divided On was intervals. induction then mussels 3-m spawning were the at mussels The containing places The performed. were night. be boxes at the would 2017, length and experiment March cm the 27 where (60 on pond box prepared the plastic to a not next inside but pond pass sand could fine water which on through placed position then of the were period spawning mussels and the stage before 2017, developmental March × larval 5 on each collected of were at pseudosericeus induction Mussels quantities. tubercles the large mussels, minute in in simultaneously the larvae of of migration development and the study To of Induction 2.2. m n lcdi h ml odweetesann xeiet ol ecnutd h captured The conducted. be would experiments spawning the where pond small the in placed and mm) 3 ± 1 ° oa f10mses(hl egh3–0m)wr olce sn iknt(ehsz 3 size (mesh net kick a using collected were mm) 35–70 length (shell mussels 150 of total A . .Antrl1:1hlgtdr htpro a sd h usl eemitie nthe in maintained were mussels The used. was photoperiod light:dark h 13:11 natural A C. .pseudosericeus R. .pseudosericeus R. .pseudosericeus R. spawning avldvlpetsaeadpsto nietemussels the inside position and stage development larval .pseudosericeus R. Chlorella × avewr eemndudraseesoi microscope stereoscopic a under determined were larvae .pseudosericeus R. 3 0 mwidth cm 100 p upnindrvdfo nido aquarium. indoor an from derived suspension sp. niiul.Tesae o a lcdi another in placed was box sealed The individuals. × 0c length cm 60 × 0c egt,wihwr transferred were which height), cm 60 .d.sinuolatus U. pwigo usl a performed was mussels on spawning × × 0c width cm 60 .pseudosericeus R. 0mhih)wsprepared, was height) m 60 usl a checked was mussels × 0c height) cm 20 ave The larvae. R. 2 . Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. ..Rltosi ewe h egtadpsto ftemnt uece n h opooia and morphological the and tubercles minute the the at parts of U position and and M facing larvae height the heads the M in their the of and had found characteristics between cavities those (U physiological suprabranchial Relationship than siphons. demibranch and inhalant faster gill demibranchs and were 3.3. the gill exhalant part the in the L to in common than the more found opposite 134) in were larvae direction larvae = larvae the the the of the The n All to space on, 92.4%, demibranch 11 time. interlamellar 11). day prevalence same the = gill from part; in n the 110); found 7.6%, (L = from were n cavity parts; migration larvae 100%, suprabranchial larval prevalence the the parts, when 10, M in day day, developmental and Until 11 per (U of larvae demibranch day observed. position the the the first after of and was position and 2), cavity the and 1, before in suprabranchial 1 (Figures Changes different Table siphon stage. were in exhalant developmental shown the stage to are through according mussel varied stage larvae the developmental the of larval of part) each ovipositor (L at spawning cavity mussels suprabranchial The the they in when 4. position 11, Figure tubercles larval day minute in until changes The vestigial The mussels EHR. remained the the they inside on and migration Larval than 6, day WLP 3.2. the on towards the of observed inclined flattened. on epidermis slightly first or higher but the were shrunken and hemispheric on became region mostly denser observed unidentifiable posterior were also were 24, the tubercles day were tubercles on minute on they No The and on, region; on. eyes. 11 27 posterior the day day from around from decreased larvae only continuously the observed tubercles were the of protuberances height small The 10. day to egtgaulyicesdfo a fe acig n h ihs auswr on nteWPo day on WLP the on found Their were hatching. values after highest immediately the observed and hatching, were development after WLP [11.4 and 1 and 7 day distribution EHR from tubercles’ the increased gradually on the height hatching, tubercles and after minute surface, Immediately hemispheric their shape. The development. vestigial on and flattened development growth tubercles and of larval minute shrunken degree with hemispheric and a many well-developed changed and hemispheric a had had shape found: of of PR were larvae were The the heights WLP tubercles the 5. and on different of EHR 4, those of the types Figures whereas surface Two PR; the and shape, regions. on and 1 among tubercles WLP, Table minute differed The in clearly EHR, vestigial-shaped. shown tubercles sites: are minute three the region in of each observed at were tubercles tubercles the minute on larvae’s tubercles The minute the The of larvae. features without gill 0.05. General different and < 3.1. among P with larvae when mussels of considered frequency of was RESULTS and significance size number 3. Statistical in the size. difference compare mussel IL, the to to test Chicago, related to performed Inc.„ parts used SPSS was was 18.0, test t-test version H sample Kruskal-Wallis (Systat two softwater SYSTAT A using USA). larvae, conducted without were and analyses with Statistical mussels of mm. size 0.01 analyses nearest the Statistical the compare 2.7. to To larvae. measured were of lengths frequency shell and mussel number the and tank. branchs the of from no removed use larva Host experiment, per of regions days the stage. three developmental of 30 larval from parts each the measured 2.6. at most were During measured and region were PR). sac heights per as yolk tubercle tubercles of minute to minute the regions referred Thirty and posterior (hereafter found, (3) were region mussels and fin-fold dead WLP), caudal as including to referred body (hereafter sac yolk ventral .pseudosericeus R. ± 2.0 .pseudosericeus R. μ 691.).Tehih ftetbrlsrpdydcesdapoiaey6%fo a 8 day from 60% approximately decreased rapidly tubercles the of height The (6.9–17.1)]. m tlzto fhs mussel host of utilization a eemndb eodn h oiino avewti h orgl demi- gill four the within larvae of position the recording by determined was .pseudosericeus R. .pseudosericeus R. 4 nee h ildmbac UadMprs or parts) M and (U demibranch gill the entered larvae Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. tti tg,tetbrlsa l ie eesalrta ntepeiu tg.O a 1 h heights the 11, day On stage. previous the in 1.7 than were smaller WLP were and sites EHR all the mussels. at on the tubercles tubercles of was the hatching) parts of after M migration stage, were 26 and components no this region day U heart At and to dorsal the 11 in their demibranchs, the (day found on the completed, stage only tubercles Reduction in were was larvae The 3.3.5. remained eyes The larvae cavity. larvae’s develop. suprabranchial The to the the began in in shortened. observed fin lens and caudal the contracted the of significantly and differentiated, development clearly the were stage, this At ntepeiu tg.O a ,tehih ftetbrlso h H n L erae o4.7 to decreased WLP and EHR the than on shorter tubercles and the smaller of drastically 4.3 height became and the WLP (1.7–3.8) 8, and day EHR On the μ the stage. on previous tubercles the hatching) the minute with after in the slightly, 10 M stage, day shrink and this to to U At 8 began in (day side found stage dorsal only reduction were Abrupt the larvae on 3.3.4. the epidermis and the observed, mussels. was and the circulation migration of yolk, larval blood parts Red the No of heads. backwards. their front lengthening under observed yolk in be and could seen heartbeat small be their very and could still eyes, were form to they began but larvae stage, The this during observed first were PR flat. the on tubercles minute Vestigial omto tg) n hi est nteERadWPwsvr ih h egt ftetbrlson tubercles the of heights The high. very the was to compared WLP 11.4 6.0 size and and were the EHR (4.2–11.0) WLP thrice the approximately and on (reaching EHR density stage the their of this at and parts highest stage), M the hatching)formation and was after U height 7 the and tubercles’ day dorsal in The to the only 6 on found (day tubercles were stage the larvae Peak and 5, 3.3.3. yolk and egg 2 the days to Between mussels. anterior the slightly developed. developed greatly head regions WLP larvae’s ventral and the EHR 4, the day On on heights small. Their very WLP. still the were and but EHR compared the size the on μ twice abundant approximately was reaching 3.3 and rapidly, were stage, very developed previous mussels. tubercles the was the minute hatching)to region of the after part caudal stage, 5 M this the day the At of to and in 2 fin-fold small found (day The were were stage which larvae identified. Growth WLP, all were 3.3.2. Moreover, of regions, pair stage. ventral this a and at dorsal but small the hatching, PR very on after the develop immediately on to Tubercles detected EHR. started were the stage. migrations on the larval those 2.6 at No were than observed WLP larger not were and tubercles. were WLP EHR minute larvae the the hemispheric the on on with of covered tubercles tubercles already the i.e., were of respectively; larvae heights (2.3–8.9), the the of 1, WLP day and On EHR hatching) the after stage, 1 this day reduction, During to reduction, hatching abrupt (from peak, stage height growth, Formation in formation, 3.3.1. changes stages: The charac- six 5). physiological into 4, and divided Figures disappearance. morphological were 1; and (Table external tubercles progressed larvae’s minute development the the larval and of as mussels related closely the were inside teristics larvae of position The 2870 n 9.0 and (2.8–7.0) m 451.)o a ,rsetvl.Vsiilmnt uece ea oapa ntepseirregion, posterior the on appear to began tubercles minute Vestigial respectively. 5, day on (4.5–12.0) m ± 1.0 μ 1956 n 5.9 and (1.9–5.6) m ± 0.6 ± ± 2.0 μ 1.5 3157,rsetvl,rahn egtsmlrt hto h omto stage. formation the of that to similar height a reaching respectively, (3.1–5.7), m μ μ 691.)o a —hnte ece hi ekhihs,respectively. heights—, peak their reached they 7—when day on (6.9–17.1) m 521.) epciey n ndy1,i ail erae o2.4 to decreased rapidly it 10, day on and respectively; (5.2–12.9), m ± 1.3 μ ± 4095 n 9.7 and (4.0–9.5) m 1.4 μ 3288 ndy2ad4.2 and 2 day on (3.2–8.8) m ± 0.3 μ 5 1226 n 3.3 and (1.2–2.6) m ± 1.8 μ 601.)o a n 6.6 and 6 day on (6.0–13.6) m ± 0.5 ± ± 0.9 μ 0.4 1841 n 4.8 and (1.8–4.1) m μ μ 2771 n 7.5 and (2.7–7.1) m 2244,respectively. (2.2–4.4), m ± ± ± 1.5 0.5 1.7 ± ± μ μ μ 0.9 1.5 m m m Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. hnegnr Km 92 97.Szk iia(94,b rpsdteeitneo he ye fyolk of types three of existence the proposed b) pseudosericeus (1984a, Hibiya R. minute and & The bitterlings, Suzuki in 1985). 1997). projections al., in 1982, et exist (Kim, Suzuki projections genera wing-like 1988b; thinae as Jeon, and & bitterlings, Suzuki in in 1984b; features 1984a, unique are Hibiya, tubercles & (Suzuki tubercles shaped whereas tubercles, minute hemispheric of the tubercles in minute remained Among that of those fact, types than in Two faster apparent; developed were clearly cavity suprabranchial ability suprabranchial the demibranches. locomotion the to the their migrated to larvae migrated regarding the when that changes when stage, individuals Thirdly, physiological reduction 6). the and 5, during (Figures morphological only M shorter cavity, occurred and became (U part) demibranchs (L and tubercles cavity the peak, minute suprabranchial of growth, the the space formation, interlamellar the to were during the migration the Second, results in larval of former. main parts); occurred the development WLP three larval in and and than stages, 5), region EHR reduction latter 4, abrupt on the Figures tubercle concentrated in 1; minute developed were (Table more mussel) stage, tubercles being host developmental minute the (larval the inside Firstly, factors larvae found. the three of among position relationship and height, the investigated study This DISCUSSION 4. was mussels the of gills outer right and inner, right inner, left outer, left the (54.05 in not larvae 1.89 of did (53.66 number that larvae The those had 127). that and mussels 85) t the between = t-test; found n were sac 38.42–68.82; length yolk shell range, the in mussels. and differences the significant open, ventral, of No were pectoral, part by anus The L utilization and the larvae. mussel mouth in Host some the only in 3.4. found developed, only were completely and Larvae were observed larvae absorbed. were completely the tubercle was of minute of fins of 91.5% caudal parts and and only respectively. 6.2%, stage, mussels, larvae) 2.3%, this the free-swimming of At of to total From part 27 remained A (day L cavity. had stage and mussels. suprabranchial that M, Disappearance the larvae the U, 3.3.6. from the in the removed tanks; found in when experimental found were freely were larvae the larvae swam in the larger the also dorsal found of slightly the mussels were Most on a the individuals those and inside of remained. free-swimming silver reduction parts yolk on, the and two by the 24 clear followed the with of day reduced, as became complete, completely parts darkened eyes side became some color anterior their Their the bladders side; and on developed. air tubercles stage, markedly their The this jaws, and front. lower at and expanded, had developed upper pigment tubercles larvae complete minute melanin the with all of heads, almost Their fins 26, brown. caudal day and On pectoral left. found. The were were them tubercles of minute traces without only larvae disappeared, some 24, day On Fgr B.Telra eesgicnl oefeun ntetootrdmbacsta nteinner the in than The demibranchs gills. outer mussels’ demibranchs two the four the the of of mussels in each parts the among frequent four 56) of = gills more and (n outer test, 44.09% significantly three, right and H were two, 5), (Kruskal-Wallis and = larvae demibranchs inner, one, (n right 3.94% The in 10), inner, 6B). = found left (n outer, (Figure were 7.87% left 56), larvae = the (n 0 in 44.09% larvae and was of 6, appearance 30, of 49, frequency of H (Kruskal-Wallis total found was A demibranchs four the among difference significant test, no 6A); (Figure respectively Acheilognathus ± = P .7(–;n=5) 1.30 56), = n (1–5; 1.17 148 .4;Fgr 6A). Figure 0.148; 0.341, = Rhodeus and ntepeetsuy uui&Jo 18)rpre httetp n opooyof morphology and type the that reported (1987) Jeon & Suzuki study. present the in P bitterlings, Tanakia .3) h ubro aveisd usl a 3.01 was mussels inside larvae of number The 0.734). = Rhodeus .pseudosericeus R. ± hywr sda aooi hrceitc odffraogacheilogna- among differ to characteristics taxonomic as used were they , .atremius R. .7(–;n=1) 1.00 10), = n (1–3; 0.67 .pseudosericeus R. P < .uyekii R. a osdrdt aetp- rjcin,wihwscnre for confirmed was which projections, type-C have to considered was .0;Fgr 6B). Figure 0.001; , and .suigensis R. larvae .pseudosericeus R. 6 avewr on:hmshrcadvestigial-shaped. and hemispheric found: were larvae ± ± 1 ) n 2.36 and 5), = n (1; 0 , .0m;4.06.6 5 w sample two 65; = n 40.10–69.96; mm; 7.20 .ocellatus R. aebt eipei n vestigial- and hemispheric both have and , Rhodeus Rhodeus ± .o smithi o. R. .7(ag,11;n= n 1–13; (range, 2.27 ± .2(–0 56), = n (1–10; 1.92 itrig u not but bitterlings itriglarvae, bitterling ± aeonly have .5mm; 6.65 Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. ta. 01 iaua 05.Temgaino avefo h nelmla pc ftedemibranchs the of space (Smith interlamellar space the et and from (Kim oxygen larvae for siphon of glochidia exhalant migration with the The compete to may opposite 2005). gills direction the Kitamura, the inside 2001; in eggs al., bitterlings’ migrate et The then 2005). opposite direction and Song, 2018). the & cavity al., in stages. Back cavity, suprabranchial suprabranchial developmental 1997; the the (Aldridge, the to in siphon over migrate exhalant they abilities that the increases, physical ability to reported that swimming gain (1994) their those Kwon they as than and & as development Song part (Davenport the slower U space 2002). sericeus than and a the Reynolds, larger supply have interlamellar is to & oxygen which the increasing to Mills return cavity, thus in found suprabranchial 1982; increased, demibranchs remained the Woolmington, be were the that to can & hatching larvae migrating rates of ventilation The By after space space, cavity. days interlamellar factor. interlamellar suprabranchial 11 limiting the The to a than larvae migrated becomes the more 1993a). and (Wu, opens, siphon for Dimock, grow shell demibranch exhalant space larvae the mussel’s & the in the the (Tankersley of occur when as must survival cavity, space expands spawning mantle larval bitterling interlamellar the therefore, exhalant increase or environment; to mussel’s cavity the to connected a to suprabranchial is enters exposed siphon the become ovipositor inhalant would in bitterling’s the The placed As are siphon. 1998). inhalant eggs one and and siphon, exhalant Further types. one their 2006b). have Kitamura, as in Mussels mussels 1985; tubercle inside Hibiya, minute migration & of with (Suzuki role compare demibranchs the to investigating hosts’ required the be of will space research interlamellar and the larger yolk in of development the fit The of 2008). al., form et the Park μ in 1990; and 1989, tubercles head, 1988d, minute the 1988c, 1988a, sharper of 1987, of part Jeon, that reduced & foremost from Suzuki abruptly different the 1985; hilly, are in or (and scaly intensively fins tubercles is in most when minute projection tubercles develop ability; the minute projection, swimming The develop, wing-like no 5). submerged to a with 4, and larvae start Figures vegetation in ability) to 1; only (Table attach swimming occur to larvae’s tubercles them have the minute studies enables consequently, nature; The that tubercles in 1980). function minute polysaccharidal attachment (Laale, be The to an or objects 2005). presumed asphyxiation perform Uehara, are by they and larvae & gill cells that Kawamura mussel bitterling epidermal shown the 2005; of unicellular in Kitamura, Mortality large death by 1994). 2000; and formed Kwon, al., mussel are & the et (Song by (Smith develop ejection WLP deficiency part premature to the nutrient factors: developed is began main during area most two WLP space, surface by interlamellar and the entered widest occurs their that on largest in larvae’s larvae growing hatched tubercles the the demibranches, The the minute time stage. was on which reduction on The settled WLP abrupt siphon fixate area. the exhalant The during mussel’s to surface shrink the harpoon. them largest through to for began a the and easy and also like hatching after it EHR, have removed, shortly the larva, making be in posteriorly, entire to those inclined the as slightly difficult of large was but speciation as tubercles 2001). gills twice on minute approximately al., the studies the were et of WLP in-depth Arai the and direction 1969; Thus, on similarities the (Mayr, study. these tubercles necessary this minute for are of hemispheric reason factors The The results specific 2015). the and of al., characteristics on those et ecological based whereas (Kim on cavity demibranches, in determined based suprabranchial the differences be the of were in cannot development space there masses Moreover, the differences interlamella however, egg 2006). during the fish; in al., yolk in laid et adult egg laid (Kin and the the are epidermis of and and the sticky morphology of projection not height the wing-like and the in area of also developmental shape the but the stages, in disappearance only and not similar very are of those study, to uyekii present R. similar the In shapes species. tubercle to minute pseudosericeus of species from types two and with time larvae over change tubercles minute these s 3–15 vs. m ave ncnrs,rmi nteitraelrsaeol uigteiiildvlpetlstages, developmental initial the during only space interlamellar the in remain contrast, in larvae, avedvlpdteetbrlstruhu oto hi R(uuie l,18) h w species two The 1985). al., et (Suzuki PR their of most throughout tubercles these developed larvae μ )i naatto taeyta lopeet rmtr jcinadalw avet tightly to larvae allows and ejection premature prevents also that strategy adaptation an is m) avehdahg ocnrto fhmshrctbrlsol nteERadWP whereas WLP, and EHR the on only tubercles hemispheric of concentration high a had larvae Acheilognathus and Rhodeus Tanakia Acheilognathus 7 itrig Fkhr ta. 92 uui&Hibiya, & Suzuki 1982; al., et (Fukuhara bitterlings avecmae otoeof those to compared larvae .rhombeus A. and .uyekii R. Tanakia Acheilognathus a eotdt ntal remain initially to reported was avewr on;however, found; were larvae itrig,wihd o have not do which bitterlings, .pseudosericeus R. Rhodeus and .signifier A. .uyekii R. .yamtsutae A. Tanakia .pseudosericeus R. ave(20–40 larvae r sticky are avefor larvae gsare eggs and larvae R. R. Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. etakD.Hu a i,D.Jn en akadD.Jn on akfrueu omnsand comments useful for declared. Park None editing. young language Jong English (R2020034). INTEREST Science for Dr. OF Fisheries (www.editage.co.kr) of and CONFLICT Editage Institute Park National thank the Seong to from Jong grant like a would by Dr. I supported was Kim, work Tae This Therefore Hyun suggestions. Dr. larvae. thank of factors. We In physiological migration role of survival. causing the advantages factors determine better to and main for ACKNOWLEDGMENTS advantages required the development mussel be evolutional of tubercles inside will determination the research minute larvae the of physiological of to of further understanding limited migration reduction was our and or investigation enhance tubercles the growth may however, minute direct study, finding the larvae provided present of this of larvae, this ejection Thus, development the premature the the of prevent hosts. characteristics of to mussel physiological developed are the their tubercles, tubercles minute and by minute of development that maturation, mussels, the evidence of inside examining comprehensive type by et larvae and study, (Smith as present of selection such this the host migration However, factors conclusion, and In various the mussels. mussels, to in of 2006). in migration Nelson, due stages eggs larval evolved 2004; laying early and al., worldwide, in position the known spawning unique lay during are type, are They tubercles spawning types and development, minute 2011). 60 time, al., called which hatching bulb-like, et structures of fast Kim types: tissue species, very 2006; four unique & a al., into develop have (Tankersley et classified development, eggs, (Kim bitterlings be larval sticky of can are of number eggs eggs small selectivity bitterlings’ some a moreover The active ovoid; history. and or life spindly, structure early pear-shape, unique regarding gill a bitterlings to have of Bitterlings selectivity related the is elucidate it to necessary 1993b). whether are Dimock, brooding and with studies demibranchs Further position (after outer 2014). June the gill al., to and For compared et May pouches (Kim observation). in brooding pouchs and without (per. demibranchs larvae) inner demibranchs the in inner identified brood mussels in spawning before analyzed, than release the (i.e., were mussels when demibranchs Interestingly, April the season) sites. and after brooding spawning March that as the in but used mussels demibranchs, be can inner of demibranchs inner in patterns outer the spawn widened reported inside bitterlings the (2003) brooding spawning larvae, Reynolds larvae, and during their their brood Mills in gills Moreover, mussels Kitamura constrained in spawning. when respectively. more group flow that gills, (Kitamura, during been sex non-marsupial total have pouches mussel and the may of non-gravid brood irrespective bitterlings that demibranchs in as female those proposed demibranchs that of (1992a) for reported outer 4% Tankersley (2006c) and competition the 16% avoid 2018). use approximately to al., that be choice et would mussels species’ Kim of these 2006c; glochidia is have 2006b, it with Studies that space suggesting ability. and no demibranchs, ejection oxygen May, outer and in after accessibility, than is ovipositor that availability, demibranchs because season reported demibranchs, space outer (2003) spawning choice, the that Reynolds the than active However, larvae reported and as more Mill reasons: had but and which unknown. four b). demibranchs, (1997) demibranchs, was inner of Aldridge 2006a, the outer mussels use study. the 2005, mainly the this bitterlings in Kitamura, during the of observed only 2004; sex was glochidia care al., the brood glochidia et so to study, Smith known present 2003; is the Dimock, 2002, & during Reynolds, (Tankersley larvae found pseudosericeus & of were speed, Mills density flow glochidia and water 1999; sexes, size, No Aldridge, as positions, such gill b; conditions among and 1993a, vary structure content gill the oxygen mussel dissolved in that and reported rate have Spence studies mortality 2006b; previous 2018). (Kitamura, larval Many al., supply lower oxygen et and increased Methling and 2013; competition growth Smith, for intraspecific & space reduce providing by may cavity cavity suprabranchial suprabranchial the to .robu,A cyanostigma A. rhombeus, A. avewr anyfudi h w ue eirnh ftefu gills. four the of demibranchs outer two the in found mainly were larvae .pseudosericeus R. and , .o kurumeus o. R. 8 a on ohv ihrsann ae nouter in rates spawning higher have to found was .signifier A. gswr on tahge aei inner in rate higher a at found were eggs wc smn avewere larvae many as twice , .d sinuolauts d. U. R. Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. i,H . o .G,Co,W . ak .Y 21) ouaineooyo oenrs bitterling, rose Korean of ecology Population (2015). Y. J. Park, & S., Ichthyology mussel W. of host Choi, Journal and G., characteristics J. uyekii development Ko, Rhodeus Embryonic S., H. (2018). Kim, Y. J. Park, Fishes & of bitterling Biology S., surface flat skin H. of the Choi, on utilization tubercles S., Minute H. (2008). Korean H. Kim, J. of Kim, History & bitterling, J., Life E. endemic Early Kang, Korean Ichthyology K., and the M. in Eggs Park, larvae Y., of of J. Development Park, H., C. (2006). Kim, H. J. Kim, yolksac & Korean) J., the E. Kang, on Bitterling, H., tubercles C. Kim, autumn-spawning scaly the in Miniature diapause free-embryonic on https://doi.org/10.1111/j.0022-1112.2005.00769.x temperature of Effects (1982). (2005) bitterling stages. K W. Uehara prolarval K, Kawamura Maekawa, in & fishes cyprinid Y., https://doi.org/10.11369/jji1950.29.232 rhodeine mussels Nagata, in of activity S., ventilatory Eu- monitoring Fukuhara, the of of patterns method of ventilatory the new introduction of A Ecology study The and a (1982). in D. use its A. (2007). and Woolmington C. & J., Smith, Davenport & R.C. Hoffimann, https://doi.org/10.1111/j.1467-2679.2007.00239.x ( N., bitterling Bogutskaya, ropean D., V. of Damme, strategy adaptation and (2000). mussel B. in N. Spawning Davies, (2005). B. H. Acheilognathinae). Song, (Cyprinidae: & M., H. Back, 8146-1 mussels. (2001). T. freshwater Ueda, Acheilognathinae). of & (Cyprinidae, R., gills S. the Jeon, R., in Arai, bitterling European of Development Biology Fish (1999). C. D. ( Aldridge, bitterling UK. of University, ecology Cambridge Reproductive Thesis, PhD (1997). C. D. Aldridge, REFERENCES 0000-0003-3189-3844 https://orcid.org/ Kim, Hyeongsu (https://figshare.com/s/10cfe2fee4bc89aeb268). ORCID figshare at available be will Data entitled is person other STATEMENT No AVAILABILITY DATA manuscript. the wrote and study this authorship. executed to and designed, conceived, H.S. Kim CONTRIBUTIONS AUTHOR celgahsrhombeus Acheilognathus hdu pseudosericeus Rhodeus 4 6-7.https://doi.org/10.1111/j.1439-0426.2007.01030.x 269-275. 24: , 2 56.https://doi.org/10.1016/0022-0981(82)90216-7 55-67. 62: , 4 3-5.hts/ doi.org/10.1111/j.1095-8649.1999.tb00618.x https:// 138-151. 54: , Pse:Ahiontia)i h ogecen akengn Rvr,Korea. (River), Mankyeonggang Bongseocheon, the in Acheilognathinae) (Pisces: 0:5-6 https://doi.org/10.1007/s10641-017-0680-4 55-66. 101: , hdu amarus Rhodeus uko,Cwid n te Cheats Other and Cowbirds Cuckoos, 7 88.(nKorean) (in 78-85. 27: , celgahsrhombeus Acheilognathus ctylgclResearch Ichthyological oenJunlo Ichthyology of Journal Korean (Acheilognathinae). 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Spence, A.S., Tarkan, Polaˇcik, M., M., Reichard, hdu sericeus Rhodeus 0 6134.https://doi.org/10.1111/j.1365-294X.2011.05198.x 3631-3643. 20: , and and oenJunlo Ichthyology of Journal Korean 8 93.https://doi.org/10.1007/s002650000212 29-35. 48: , :72.https://doi.org/10.1007/BF01103363 7-22. 5: , Pseudoperilampus hdu suigensis Rhodeus hdu sericeus Rhodeus ). rceig fteRylSceyo odnSre B Series London of Society Royal the of Proceedings ora fZoology of Journal (Cyprinidae). 1 9-0.https://doi.org/10.11369/jji1950.31.198 198-202. 31: , (Cyprinidae). ). hsu amarus Rhoseus h ot fprna aei ees fishes teleost in care parental of costs The rswtrBiology Freshwater :3-0 i Korean) (in 39-50. 6: , caZoologica Acta 6:1714 https://doi.org/10.1017/S0952836903004497 107-124. 262: , hdu ocellatus Rhodeus aaii id n hi Hosts their and Birds Parasitic Oikos aaeeJunlo Ichthyology of Journal Japanese 11 aaeeJunlo Ichthyology of Journal Japanese ,afihta aaiie rswtrmussels. freshwater parasitizes that fish a ), 83:5955 https://doi.org/10.2307/3545615 539-545. 78(3): , 4 1-1.https://doi.org/10.1111/j.1463- 113-118. 94: , 6 0-1.https://doi.org/10.1046/j.1365- 903-911. 46: , hdu sericeus Rhodeus celgahsyamatsutae Acheilognathus . ra,E,Odacoa . mt,C. Smith, & Ondraˇckova, O., E., Ercan, O., ¨ celgahsyamatsutae Acheilognathus mro aaiiefehae mussels freshwater parasitize embryos ) oenJunlo Limnology of Journal Korean ). vltoayEcology Evolutionary . tde nCoevolution. in Studies : eiw nFs Biology Fish in Reviews eairlEooyand Ecology Behavioral Rhodeus 6:1327-1334. 267: , 2 335-344. 32: , Cpiia)into (Cyprinidae) 1 287-296. 31: , (Cyprinidae). (Cyprinidae), Molecular Evolution Acheilog- Rhodeus 31: , Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. Data may be preliminary. asatrhatching after Days Big in Unionidae) (Mollusca: pseudosericeus mussels ( lampsiline Mean of 1. species Table four of host Fish Virginina. Creek, (1982). institue R. Mocasin Thesis, Neves, PhD & river. A.V., China. Changjiang Wuhan, Zale, of sciences, reaches of mid-lower academy in Chinese Mollusca hydrobiology, freshwater of on Studies (1998) X Wu parasites. Press. 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Akiyama, skin bitterling, N., the the Suzuki, on of tubercles Development minute (1990). on R. note S. a Jeon, surface. & skin bitterling, N., the the Suzuki, on of tubercles Development minute (1989). on R. note S. a Jeon, & N., in surface. Suzuki, implication skin the genetic on the tubercles bitterling, on minute the on Korean). and of note Development surface a (1988d). skin with R. Korea, the S. Jeon, on & tubercles N., Suzuki, minute on surface. bitterling, note skin the a of the embryos. Development with hybrid on (1988c). Japan, tubercles R. and of S. minute Korea juveniles Jeon, on and & note larvae N., Suzuki, a eggs, with of Development Cyprinidae), Limnology of (1988b). (Pisecs: Journal R. Korea S. River, Jeon, Ansung & surface. skin N., the bitterling, Suzuki, on the of tubercles Development minute on (1988a). R. note S. a Jeon, & N., Suzuki, larvae ± oenJunlo Limnology of Journal Korean D egto iuetbrl ntesraeo w ein n hrceitc of characteristics and regions two of surface the on tubercle minute of height SD) 1 -5 i Korean). (in 1-15. 21: , stage Developmental aainJunlo Zoology of Journal Canadian Coevolution yaoo cataracta Pyganodon yaoo cataracta Pyganodon nMPgl d nylpei feouin xod Oxford Oxford, evolution. of Encyclopaedia ed. 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Data may be preliminary. 7Dsperne--alkn ffins of kind all - 0.11 0.26 0.38 - 0.56 0.72 0.84 Disappearance 0.87 0.95 0.98 1.01 1.07 1.13 1.28 27 1.42 26 25 1.61 24 23 22 21 20 2.43 19 18 3.51 17 1.72 16 15 14 13 Reduction 6.62 12 4.67 4.22 reduction Abrupt 6.05 3.83 3.57 11 10 9 Peak 3.26 8 7 2.57 Growth 6 5 Formation 4 3 2 1 hatching after Days stage Developmental uece (um) tubercles minute of Height ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± .60.19 0.47 0.65 0.16 1.03 0.20 1.19 1.25 1.41 0.29 1.41 0.13 1.45 0.16 1.57 0.16 1.70 0.20 1.78 0.17 2.08 0.16 0.19 2.51 0.17 0.19 3.09 0.20 0.24 0.23 3.27 4.28 6.24 0.30 8.95 0.46 0.75 11.42 0.88 9.68 7.45 1.53 6.80 6.27 1.26 0.90 5.91 1.15 0.82 4.85 0.99 0.51 13 uece (um) tubercles minute of Height ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± .0free-floating 0.29 0.38 0.50 0.28 0.29 0.29 0.26 0.29 0.28 divide bladder 0.35 air 0.54 and 0.49 pectoral 0.51 0.43 0.51 were Larvae complete lens begin fin caudal tubercle 0.40 minute 0.58 1.02 1.51 form, eyes slightly head 1.76 to start 1.52 larvae 1.83 1.71 of movement no 1.38 1.68 .4rdbo circulation bood red 2.04 complet shrink yolksac ventral and dorsal confrim larave of Characteristics appear individuals rooms two into develop fin caudal reduce completely tubercle minute cavity suprabranchial the in observed develop to reduce and contract observes heartbeat develop movement regions ventral and dorsal in develop to begin yolksac larvae Posted on Authorea 10 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158137013.34385109 — This a preprint and has not been peer reviewed. 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