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Research Article Horizontal transmission of the ectoparasite arcuatus (: ) to the next generation of the three-spined stickleback Gasterosteus aculeatus

Jaakko LummeE and ;

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In the parthenogenetic monogeneans of the genus Gyrodactylus HE15;7!'7@' hosts is determined by the relative roles of lateral transmission and clonal propagation. Clonality and limited transmission lead to 7!73!!B773%@%Gasterosteus aculeatus Linnaeus, the local mitochondrial diversity of Gyrodactylus arcuatus7'%E-557!7ƽ3 '%K7!77>! %@%'!7@%7'M!NNNcox1513 77'[B!7!7'7!777 (h) was >ƽ@'[7'!!@Q hR2-;<h = 2-51SBG@9

K7!QEM9S7FF*FF;2E13<+322<

Some important aspects of the population genetics and ten overcome by having a very large number of random3 ƽ ly spread propagules. The main characteristics of the host 7 3! ! K7 3 which determine the metapopulation structure and the namics among demes on single hosts in many parasite strategy of the parasite are: (i) the carrying capacity of !@>7M !777*=QS7!3 of host generations and randomness in the transition pro3 ity of the host, relative to the turnover rate of the parasite cess. The hosts represent ephemeral resource units. If the Q7S=QS7 parasite is not vertically transmitted, the new host genera3 77=QS776 tion is born uninfected, and the parasites have – actively or factors which determine metapopulation structure and par3 passively – to locate and infect new host individuals. The asite strategy include the dispersal capacity and length of death of a host means the end of the resource patch. The the dispersal phase of the parasite. The parasites often have transition between host generations is critical, and the life 7>@'>! cycle of the parasite should be adapted to this transition. The simple and direct transmission biology of hyper3 7'773 viviparous, parthenogenetic and hermaphroditic mono3 3!%7[3 ! \' 7 ! Gyrodactylus Nordmann, !F@Q7ZE-<9S E15;''@!. Q;22+S' K77![!@'73 D755%%Q;22NSGyrodactylus para3

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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. E2E99EEF;2E122< Z]*Gyrodactylus arcuatus

@'3 K7xV%%Gyrodactylus arcuatus 3 Q%%;22;;22NSK7!73 7'%E-55751cox1 @ 3!' 77ƽ@'xV%% QD@];22;SK77 H;N2%'!!@

7[7 QST R22EEZ;2E<@SK7ƽ@3 K7!7!373 served between G. salaris and G. arcuatus or G. gondae distinct phases for dispersal or dormancy but, at any time @ > @ ƽ! 7 3 during their life, the worms are able to switch from one @ 7 ƽ! 77Q!;22+S 73! G 7 ! M 7 3 ]'@ tance of transmission between host individuals and/or the transmission of G. arcuatus7>! generations, and the clonal propagation of gyrodactylids. host in the wild. In the brackish water coastal habitats of In the Gyrodactylus model, the transmission is direct, but 7773%@%QGasterosteus @M 7! ! aculeatus Linnaeus) forms dense and continuous popula3 [ 7!7 3 QD;221;2E5SB[ M ! parasite G. arcuatus is also common and numerous, as well > Malmberg, @!!Q;221Z E-+NSalmo salar Linnae3 ;2E<@S77!77 us, the populations were strongly structured, apparently by of the parasite, transmission can be studied at the level of !Q_;22-SB77 7& ' 7' 7 7 @' spawning rivers in the great lake basins of Onega host generations may present a model for studying the pop3 and Ladoga in Russian Karelia, only one parasite mito3 ulation dynamics of the parasite, which is important for un3 chondrial haplotype was observed. There is no transmis3 !77733 sion between adults feeding in the open lakes. The parasite K773%@%@ @V77 !Q!7;222@;2E5S 7@ƽ Studies of the parasites, which have a shorter generation B7!Kq77 time, might also add an interesting dimension to the under3 parasite G. salaris[77 !7@!77QD;22+= distributed unevenly in the upper and lower parts of the ];22+=Z;2E<@S 9<2%!!K73

ƽ'FSTR2+59QZ;2E<S MATERIALS AND METHODS G. salaris55U7VQS The parasite material investigated here was partly utilised for 1;U73!!VQSK7 other purposes earlier. The global population structure and phy3 parr populations were apparently not infected via the mi3 logeography of Gyrodactylus arcuatus was previously reported grating adults or smolts, but the infection was maintained QZ;2E<@SB77xV%%' in the nursery rapids via transmission between sedentary V'7! V!2M9QZ;2E<SK7 hosts. The details of the genetic structure among old and young '77[Q3 777>!3 ;221SK7!77 eration of sticklebacks is reported here. Knowing the details also 7>7! provides an important lesson for parasite sampling: fewer adults was limited even in the crowded smolt trap (Lumme et al. 7V7!7 ;2E<SK77!ƽ3 local genetic diversity. tion is maintained by local coadaptation of the host and 7![777 ! lower Tornio River. %@%Q[77GZ!7+NM @ 7!7 B !7>@@ samples around the North Sea, the cox2 of Gyrodactylus 7>77 DE1;- gondae]@!x%;2297!@ The parasite load included thousands of Gyrodactylus covering host Pomatoschistus minutus Q6S 7' 7 77[7Q!ES7 diversity (hS@'2<;92N197@ Schistocephalus solidusQENN

6!;2E1<+22< 6!;1 E2E99EEF;2E122< Z]*Gyrodactylus arcuatus

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10 mm H ' @ !! ! E2)@ƽQE&6Dq@ƽ29+UQFSK';229+U QFSH692<23!F_SK7@'@3 5 mm <+ D;+'_!7 -+ DE277 9 DM;37' 6Dq[ K7;2)6Dq>E&

6Dq@ƽ2;HK6;!D;E)7 2+"KMHQxZ7S ;)!K7!['-9 D5 51-9 D for 40 s, +2 D52N; DE+ [>N; DN. K7 BK H % 7 [ ' [ '7 BKE Q+ˆ3KKKDD#K##K# DDK35ˆSBK;qQ+ˆ3##KKDD#DKK#KD35ˆS ' M '7 BKEq Q+ˆ3KKK#D#KKD###D3 D#35ˆS BK; Q+ˆ3K##K##KDDKD##DKD35ˆS3 Fig. 1.6 Gasterosteus aculeatus Linnaeus H;[7'!7!@Gyrodac- [Q;221SK7 tylus arcuatus7'%E-55'!@7 M'#%Q99+-9;S >! 7 H Q‰E<22 ! cox1) was am3 [ '7 D>< Q+ˆ3KK##KDK#D#DK##KK35ˆS E<q Q+ˆ3DKKKKDK#K#D##35ˆS 3 H;N2%7!7''77 Q;2E2SK7ZE xV%% Q+ˆ3KK######KKK##K35ˆS D>N Q+ˆ3KKKKDK3 K7 [ V [7 Q[7 7 G"H# !7 ##KK##D#K35ˆS qD>; Q+ˆ3K##DK##3 E1M;NHR<-S'77 D#DDD35ˆS'M!K7M' xV%%[7'%-

;9V!7E-M;NB7 Composite Likelihood distances (dMCL), correcting upwards the 7'9;U large distances reaching saturation. The bootstrap test was con3 B7Q+S7'3 '7E222' M7[7Q5+!7S@7V [ ! QN5 !7S ' [ @ 7 NNN77QZ;2E<@S " &7 The randomness of the numeric parasite distributions on Q7R>! 7 V [7 ' '7 6 @ Q;17R@@!7>Q Q>R;RRS@3F = s;F>

7;1R dfER3E;R.K7@7' ; 7RHFQH3ESŽQE3 SK7[>>FST based

"# 7M'FST = (hT-hS)/hT, where

K7@!7NNN!7H hT is the total diversity and hS is the mean diversity in separate was conducted and the global mitochondrial variation was report3 K7ZST'777ƽ QZ;2E<@SK77!77 was estimated by ǓǍdžǒǖNJǏ5EK7@73 xV%%[EK7[ lotype diversity (hSQ4S 7xV%%7[;#%3 QKVRDRFs) were also calculated using ǓǍdžǒǖNJǏ5E @MG. arcuatusQ‚2N1N2EM Q>ƾ;22+S ‚2N1NE-"1<;<1EM"1<;N29SG. lucii Kulakovskaya, K7>@73 E-+;Q99

6!;2E1<+22< 6!51 E2E99EEF;2E122< Z]*Gyrodactylus arcuatus

Table 1.]Gyrodactylus arcuatus7'% arisQ77!77!S@ƽ7 E-55xV%% of G. lucii. BK@;7H77[73 ] GZ G"H# G'7GZ E E 3 77[7'@G. arcuatus ; < N A4 E 5 Q!ES7'@ N E 3 sample for global phylogeographic analysis. 1 E 3 The parasite sample separately counted from the seven - N 1 [7;+!77 E2 3 ; ' 7 ! [7 7 EE E 3 E; E 3 7![7K7 E9 E 3 GZ E- 7 E+ '77 ' 3 E+ 3 E gletons (present as one individual). The other four hap3 E< E 3 ' @ ; 9 < N EN E 3 7[7QK@;S@3 E1 4 E9 E- E E QHR;2=Z;2E<@S[7H ;5 3 5 ;N2 % 7 ! 7 E1 ! ;9 3 ; and only one haplotype pair. The most numerous haplo3 ;+ 3 4 ;Q[7S-Q[[7SE1Q ;N 3 E [7S'@7xV%%H ;- 3 - 52 3 E '77'!ƽ@7 5E 3 E QSTR22EE=Z;2E<@S 5; 3 E An unbiased haplotype diversity estimate, compen3 5+ 3 E !7ƽ*h RQHFQH3ESQE3;), 51 3 E was h R2-;<77GZh R2-E<7 5- 3 E G"H#K7Gx@'GZ A40 3 E 9< 3 E G"H#!7'@![ƽ3

5 E 3 tion ZST R2252N+Q6R22;N25/22E-9EE23 DE 3 ; SM77!77 D; 3 E [7'@@7 D5 ; 3 '&77V!73 C4 3 ; D+ E 3 fection from the common pool of parasites, but this occurs D< E 3 @K7!>!@ DN E 3 in highly variable populations. D1 3 E B7[VQ;G@;229S3 DE2 E 3 [7 E5 ' G ' 3 K51 59 <- @Q79[7HE;SK777 #% @ ‚2N1N2EM‚2N1NE- "1<;<1EM 'ƽ7[7HEN7 "1<;<-9"1<;<-<"1<;<--M"1<;N2;H773 ' ƽ 7 G@! ' 3 QDSZQ;2E<@S 7!77[7'@7 7 [7 @ ! !7@Gx@'!77' a common pool with haplotype diversity H R 2-;< KR225E5Uƽ@'77 6 R QE32-;

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PW'1$QW

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Fig. 2. Comparison of Gyrodactylus arcuatus7'%E-5577xV%%'7!@73 lotype diversity of Gyrodactylus lucii_%%E-+;Gyrodactylus salaris @!E-+NK7!]E-1<!Poecilia reticulata6M6K77!77!QS 7!H@@@E222

Table 2. Clonal diversity of Gyrodactylus arcuatus7'%E-557QGZS'7V QG"H#S%@%xV%%77

7 ] N Nh h mean h GZ HE E;E1 5 5 E222 ;<21;229 H; ;9E1;&D5 + 4 E222 H5 ;&;;&-D+D< < 4 21

HM@=HhM@7=7M7

7 > ' 7 7 Table 3.HKVRDRFS in parasites on GZG"H#[77 had immediately started propagation on the new host, mul3 tiplying the number of their mitochondrial haplotype. Most Statistics GZ G"H# probably, this increase in numbers is clonal, but the alter3 KVRD test Sample size 59 <- 77!>@ S (variable sites) 9; 91 @ V G 7 7 7 7 V [7 H 95 4QƽS <212 9;N1 KVRD 3E9N< 3E1-2 [7! 6QD+2S 22<+ 2221 7@>@7Q6R299S 7! RFS test No. of alleles E1 ;9 4 <212 9;N1 QK@5SKVRD'!![ > EE-29 E;<99 7G"H# 7 QD R 3E1-2 6 R 2221S R FS F 39;;1 31992 S '!![77QF R3199 6QF +2S 22<+ 222; S S 6R222;SK7>@@73

6!;2E1<+22< 6!+1 E2E99EEF;2E122< Z]*Gyrodactylus arcuatus

P = 443718/106 P = 151/106

P = 1348/106 Number of mtDNA clones Number of mtDNA

P = 0/106

Fig. 3.K7HGyrodactylus arcuatus7'%E-557Q!S 7V773%@%K7@%7@@H 77@7VK7777>@ Q/S'7M@7517xV%%K767 dot indicate the probability of deviation from the null hypothesis. Open dots represent the accumulation of haplotypes when counted [7HEM+QK@;S

R'7GZ H'77K77 7 EE- 7 ' > @ E1 ' @3 of the propagation of G. arcuatus has still to be taken as '77G"H#77'7!7 @ 77 H QS E;<>vs ;9@73 @>7@[! xV%%7'7 @!@7> present than predicted by the neutral model based on the 7@7![7 molecular indices variable nucleotide sites (S) and nucle3 individuals is parthenogenetic propagation. This prediction Q4S]'7[77 @ 7 QES > G"H#77'7'73 reproduction should normally prevail in a hyperviviparous ' 7 > @ ! !@!Q;S[!7@ from the common pool. 7! ! M 7 ƽ! Q5S7@%7> DISCUSSION an initial colony. $!%& The local vs !@ ! ƽ It might be of interest to point out that the capacity for G. arcuatus and G. salaris Q _ ;22N clonal propagation of species of Gyrodactylus is usually Z;2E<@SK7!@! % ! @ > ] @7@7Q!;SB7 QE--1S7![3 molecular evolution were similar in both species it would ality of G. gasterostei#E-N9@!7 suggest that they have accumulated mutations in mito3 E+!M 7 H @% This species belongs to the wageneri group and is not ]'7!!773[@ closely related to G. arcuatus. D@ ] Q;22;S 7ƽ7'3 considered the clonality as an option to be used in the be3 cies. In G. arcuatus 7 ginning of infection in a new host, but they also supposed >@ƽ!7 7>ƽ7 conclusion that the total parasite population is very large life cycle of species of Gyrodactylus in general. A lack of '3QZ;2E<@SBG. salaris, suitable markers has so far prevented serious testing of each local freshwater population is nearly monomorphic, 777Q%%;22NS @7ƽ7!7 !77H 777!73 markers, the importance of clonality was obvious in G. sal- tic or in the large lakes, parasite transmission does not aris on wild populations in the Tornio happen among them in feeding grounds and therefore they q7@QZ;2E<SG are not transferred between the nursery rapids (Lumme et genetic marker used in the present study was mitochon3 ;2E+;2E<S

6!;2E1<+22< 6!<1 E2E99EEF;2E122< Z]*Gyrodactylus arcuatus

K7G. arcuatus are very derived from earlier ecological literature on stickleback ƽ D Q;221S Qq%;221;2EE*! '% ! ƽ ! 7 7 ;22-DD7;2EEqV;2EES among the females, which then may disperse better. Of The host response to the ectoparasite load should be of course, the parasite takes advantage of the most dispersing 7!77E+2 >!!K777 G. alexanderi*_%E- Q_;22-Z;2E<S]'3 6 7 ! '7 7 77!773 7Qx!' %@%Q!ES!7@73 ;22-S7!77'77 !77>! orientation and attachment of the parasite, crucial to the 7!!7\7[7[7 › ! 3 7'77!@3 tectable would be a good strategy for the host, and the par3 7 7 73 %@% '77 77'&7%@%V parasite transmission between adults, might be the key to 77@7›D7@[ 7[7M paternal care compensate for the risk of becoming infect3 the nest, and delivers sperm on the eggs immediately, the ›K7VQMS%73 >7! ! 7 7 ![7@!!3 whole breeding population must be continuous, and may Q!S@7[7 7 M 7 QKE-<5SK73!G. alexanderi

[7QFSTR22SK7![7 released from G. aculeatus was lowered due to predation ƽ>B7 @V%@%77@% @>! caused by the worms, but predation might also increase the risk of G. arcuatus7@Q>3 @!QZE-N9SV3 S7'7 7q6!Q7S 7G"H#7[7'7% G. arcuatus'7!%@%Q V'7@@! ;221S@7Gyrodactylus worms This process deserves detailed empirical investigation. '77%%@%V› K7 ! M 7 basis of the present results, as well as from what can be K7 ' @K7 7 Q!<5N1NE59+-;ŠZS

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ǏǕǕNJǍǂ6qǐǎǂnjnjǂǏNJdžǎNJ_ǖǖǔdžǍǂŠ_ǐǔnjNJ6;2213 DǓNJǔDŽNJǐǏdžD6ǐǖǍNJǏqǍǐǖNJǏ;22+3 idemiology of Gyrodactylus salaris (Monogenea) in the River ogy of parasites: elucidating ecological and microevolutionary KV%'Š75E5N5M51; E9;;9NM;;+N ǂnjnjdžKDǂǃǍdžŠ]ǂǓǓNJǔ6;22NK7@!#3 džǂǗdžǓNJŠŠǐǏǔǔǐǏ6qdžǓNJǍǧŠ;2E5]! !K7Oq3_P63 ! ƽ 7 %@% 3 <9E

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_ǖǖǔdžǍǂŠ]ǐǍǐǑǂNJǏdžǏqdžNJǏNJǍǧǏǕǕNJǍǂ6_ǐǔnjNJ qǂdžǚǎǂdžnjdžǓǔ Š &džLjǏdžǓ _ ]ǖǚǔdž K xǐǍDŽnjǂdžǓǕ 6 NJȉǕǂǓǂ xdžǔdžǍǐǗ 6ǓNJǎǎdžǓ Dq Zǖǎǎdž Š ;2EE B 7 ! ;22-DGyrodactylus salaris Gyrodactylus gasterostei on sympatric and allopatric popula3 !QSalmo 7 73 %@% Gasterosteus aculeatus. salarS9<;EM55 6+1;NM59 _ǖǖǔdžǍǂŠNJȉǕǂǓǂZǖǎǎdžŠ;22N]@!3 DždžqǐNJNjŠ]ǂǓǓNJǔ6ǂDŽDǐǍǍD;2EE!3 3[ Gyrodactylus salaris: a model for !\'!73%@% @7'77! ;+;ENM;;< E<+;59M+;9+ DŽljǍǖǕdžǓ ;222K7!qG> ZdžǔǕdžǓqŠ#DžǂǎǔŠqE-N9Gyrodactylus alexanderi: repro3 "6H'%;-< ƽ7Gasterosteus aculeatus. KǂǎǖǓǂ_ǖDžǍdžǚŠHdžNJ_ǖǎǂǓ;22N#93 DŠ+;1;NM155 #Q#S'x3 ZǖǎǎdžŠǏǕǕNJǍǂ6qNJǏǕǂǎǧnjNJ6_ǐǔnjNJ6qǐǎǂnjnjǂǏNJdžǎNJ 92;9E+-

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