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Mo phopa ology and gill eco e y o A lan ic salmon du ing
he pa asi ic de achmen o Ma ga i i e a ma ga i i e a
Jou nal:
Jou nal o Fish Diseases
Manusc ip ID
JFD-2021-44
Wiley - Manusc ip ype:
Resea ch A icle
Da e Submi ed by he
Au ho :
03-Feb-2021
Comple e Lis o Au ho s:
Cas illo, Ped o A.; Uni e sidade de San iago de Compos ela
Va ela-Dopico, Ca uxa; Uni e sidade de San iago de Compos ela
Be múdez, Robe o; Uni e sidade de San iago de Compos ela
Ondina, Paz; Uni e sidade de San iago de Compos ela
Qui oga, Mª Isabel; Uni e sidade de San iago de Compos ela
Keywo ds:
Keywo ds:
gill healing, pa asi e de achmen , <i>Salmo sala </i>, glochidiosis,
F eshwa e Pea l Mussel
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1Mo phopa ology and gill eco e y o A lan ic salmon du ing he pa asi ic de achmen o
2Ma ga i i e a ma ga i i e a
3Sho unning i le: Gill eco e y a e he pa asi e de achmen
4Cas illo, Ped o A.1; Va ela-Dopico, Ca uxa2; Be múdez, Robe o1,3; Ondina, Paz2; Qui oga,
5Ma ía Isabel1,3
61. Depa men o Ana omy, Animal P oduc ion and Ve e ina y Clinical Sciences, Facul y o
7Ve e ina y, Uni e sidade de San iago de Compos ela, Lugo, Spain.
82. Depa men o Zoology, Gene ics and Physical An h opology, Facul y o Ve e ina y,
9Uni e sidade de San iago de Compos ela, Lugo, Spain.
10 3. Ins i u o de Acuicul u a, Uni e sidade de San iago de Compos ela, 15705, San iago de
11 Compos ela, Spain.
12 ORCID: Ped o A. Cas illo: 0000-0001-5499-7190; Ca uxa Va ela-Dopico: 0000-0002-
13 3513-1444; Robe o Be múdez Pose: 0000-0003-4969-4122; Paz Ondina: 0000-0003-4392-9250;
14 Ma ía Isabel Qui oga: 0000-0001-6832-7665
15 Co espondence
16 Robe o Be múdez, Depa men o Ana omy, Animal P oduc ion and Ve e ina y Clinical
17 Sciences, Facul y o Ve e ina y, Uni e sidade de San iago de Compos ela, Campus Uni e si a io
18 s/n, 27002, Lugo, Spain. Email: [email p o ec ed]
19 Acknowledgmen s
20 The au ho s would like o hank he assis ance o D . R. Masca o and R. Ocha an wi h he
21 ield wo k and he cap i i y p ocedu es du ing his long- e m expe imen al ial. Also, we wa mly
22 hanks S. Macei as o he ho ough his opa hological echnical assis ance and D . A. M. de
23 Aze edo, D . A. P. Losada and D . P. Ronza o he scien i ic ad ices. The conse a ion
24 p og amme was co unded by he “Fundación Biodi e sidad” wi hin he Ma MaCul and
25 Ma gaSalmo P ojec s and by he Xun a de Galicia wi hin “P og ama de Consolidación e
26 Es uc u ación de Unidades de In es igación Compe i i as” (ED4313 2019/24 and ED431D
27 2017/22) o he egional de elopmen o scien i ic ne wo k. P. A. Cas illo held a Uni e si y
28 P o esso ship Fo ma ion (FPU) g an om he Spanish Minis y o Educa ion, Cul u e and Spo
29 (FPU17/02004). We also acknowledge he suppo o he esea ch collabo a ion ag eemen wi h
30 he Conselle ía de Medio Ambien e, Te i o io y Vi ienda (Xun a de Galicia).
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31 Da a a ailabili y s a emen
32 The da a ha suppo he indings o his s udy a e a ailable om he co esponding au ho
33 upon easonable eques .
34 Con lic o in e es s a emen
35 The au ho s decla e ha hey ha e no compe ing in e es s.
36 Abs ac
37 Du ing he conse a ion aquacul u e o he eshwa e mussel Ma ga i i e a ma ga i i e a,
38 ish heal h has become a conce n due he need o mussel la ae (glochidia) o pa asi ize he
39 salmonid gills and me amo phose in o ju eniles. Howe e , he e is lack o in o ma ion abou he
40 impac on ish du ing he ju enile de achmen and he subsequen gill healing. To e alua e he
41 mo phopa hological changes and gill eco e y a e he pa asi ism o M. ma ga i i e a, i y-one
42 A lan ic salmon y (Salmo sala ), in es ed wi h a ound 22 la ae/ ish g, we e nec opsied du ing
43 he synch onized de achmen o he mussel ju eniles, and gills we e assessed by
44 s e eomic oscopy, and by ligh and scanning elec on mic oscopy. Salmon showed no clinical
45 signs du ing he ial and gills eco e ed hei no mal mo phology almos comple ely in a sho
46 ime, sugges ing a minimal impac on ish heal h a e glochidiosis. In his sense, he non-e osi e
47 d ople de achmen and he goble cell hype plasia a o ed an e ec i e gill emodeling media ed
48 by apop osis, pola iza ion and cell shedding o he gill epi helia, p o iding insigh s o he de ense,
49 clea ing and healing mechanisms o he gill. These mo phopa hological echniques could also be
50 implemen ed o p ese e ish wel a e and o op imize he a i icial b eeding p og ammes o
51 endange ed eshwa e mussels.
52 Keywo ds
53 gill healing, F eshwa e Pea l Mussel, ish pa hology, pa asi e de achmen , Salmo sala ,
54 glochidiosis
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55 1 In oduc ion
56 F eshwa e mussels a e bi al es wi h ex ao dina y capaci ies o bio il a ion and bu owing,
57 hus, hey p o ide signi ican ecosys em se ices and also indi ec ly p o ec many o he species
58 (Vaughn, 2017). Despi e i s impo ance, ce ain naiads as Ma ga i i e a ma ga i i e a (L., 1758)
59 a e ca ego ized in he Eu opean Red Lis o IUCN as C i ically Endange ed due o he se ious
60 decline o he popula ions (Cu elod, Seddon & Neube , 2011). To p o ec he mos a ec ed
61 popula ions, which lack na u al ec ui men (Lois, Ondina, Ou ei o, Ama o & San Miguel, 2014),
62 one eme gency conse a ion s a egy is o implemen a i icial b eeding p og ammes (Gum,
63 Lange & Geis , 2011).
64 The cul u ing echniques o eshwa e mussels depend on he compulso y la al pa asi ic
65 s age on A lan ic salmon (Salmo sala L.) and b own ou (Salmo u a L.) y, ega ded as a
66 mechanism o dispe sal, nu i ion and p o ec ion (Ba nha , Haag & Ros on, 2008; Denic,
67 Taeube & Geis , 2015; Geis , 2010). In pa icula , he la a o M. ma ga i i e a clamps and
68 encys s in o he salmonid gills o se e al mon hs un il i de aches in sp ing––once empe a u e
69 ises o e 15 ºC––as a ee-li ing ju enile (H uska, 1992; Taeube , Gum & Geis , 2013).
70 Acco dingly, ish su e a mul i ocal p oli e a i e b anchi is, which leads o a disease s a us
71 known as glochidiosis, gi en by he name o he in es ing la ae, he glochidium (Ka na &
72 Millemann, 1978).
73 This hos -pa asi e in e ac ion supposes a bo leneck o eshwa e mussel aquacul u e and a
74 conce n owa ds he wel a e o he hos ish. In an a emp o op imize he cul u ing e o s se e al
75 s udies had es ablished ce ain ecommended glochidial loads (Taeube & Geis , 2013), based on
76 he impac on ish su i al, g ow h, espi a ion, me abolism, swimming capaci y and ish
77 beha io (Chowdhu y, Ma jomäki & Taskinen, 2019; Cunjak & McGladde y, 1991; Filipsson,
78 B ijs, Näslund, Wengs öm, Adamsson e al., 2017; Ös e ling, Fe m & Piccolo, 2014; P es on,
79 Keys & Robe s, 2007; Thomas, Taylo & Ga cia de Leaniz, 2013). Howe e , he e is a lack o
80 da a abou he pa hogenesis o he disease, in which he mo phopa hological e alua ion o he gill
81 lesions emain o e looked, mos ly ocused on he p ema u e ejec ion o un iable la ae, which
82 is associa ed o an e osi e b anchi is du ing ea ly s ages o glochidiosis (Cas illo, Va ela-Dopico,
83 Ondina, Qui oga & Be múdez, 2020). This con as wi h he ex ensi e knowledge o o he gill
84 ec opa asi es which comp omise he ish wel a e in aquacul u e, e.g., Neopa amoeba pe u ans
85 and Ich hyoph hi ius mul i iliis (Powell, Lee , Robe s & Jones, 2008; Tumbol, Powell & Nowak,
86 2001). Las ly, gills a e able o eco e a e emo al o he inci ing cause as chemicals o
87 in ec ious agen s (Daous & Fe guson, 1986; Kudo & Kimu a, 1983; Sales, San os, Rizzo,
88 Ribei o, San os e al., 2017; Spea e, Ca ajal & Ho ney, 1999); ne e heless, e y ew s udies
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89 e e o he eco e y o lesions a e glochidiosis (Kaise , 2005; Ka na & Millemann, 1978;
90 Scha sack, 1994; T easu e & Tu nbull, 2000).
91 Employing his a i icial glochidiosis as a model o pa asi ic gill disease and eco e y in
92 A lan ic salmon, he pu pose o his s udy was o pe o m a comp ehensi e mo phopa hological
93 e alua ion du ing he la e s ages o glochidiosis including he ju enile de achmen and he
94 subsequen gill healing p ocess. Hence, his s udy may also p o ide insigh s o unde s and his
95 complex hos -pa asi e in e ac ion and o p ese e ish wel a e du ing he ea ing o hese
96 impo an and endange ed eshwa e mussels.
97 2 MATERIAL AND METHODS
98 2.1 Expe imen al gill in es a ion and selec ion o in es ed ish
99 Expe imen al ish came om an a i icial glochidiosis ial pe o med in Sep embe 2015
100 (Cas illo e al., 2020). B ie ly, 1000 salmon y we e exposed by ba h imme sion o M.
101 ma ga i i e a glochidia (1,000 glochidia / ish g am) collec ed om g a id wild mussels. A g oup
102 o non-exposed ish se ed as con ol g oup. A day 14 pos -exposu e (PE) an ea ly de achmen
103 o in iable glochidia occu ed and success ully in ec ed ish could be only iden i ied by means o
104 ligh mic oscopy, due o he la al size a his s age (diame e o 70 μm).
105 A e his ea ly ejec ion, he emaining salmon we e ea ed o six mo e mon hs o allow he
106 de elopmen o he encys ed la ae and became mac oscopically isible on he gill (diame e o
107 350 μm). Thus, a e 202 days pos exposu e (PE), each exposed ish could be in i o diagnosed
108 as in es ed o non-in es ed by manual immobiliza ion, abduc ion o he ope cula and gill
109 isualiza ion. This diagnos ic p ocedu e was pe o med in less han ou seconds. As a esul , 51
110 ou o 600 exposed ish we e selec ed as in es ed (p e alence o 8.5%). The same explo a o y
111 p ocedu e was pe o med on 51 non-exposed ish. Mo eo e , o ensu e an op imal ish heal h
112 s a us and con i m he absence o any o he pa hological p ocesses p io he in i o p ocedu e,
113 i e exposed and non-exposed ish we e nec opsied and p ocessed o his opa hology.
114 2.3 Synch oniza ion o ju enile de achmen
115 In es ed and con ol ish (n = 51, mean weigh 5.8 g and leng h 8.1 cm) we e eloca ed in o a
116 eci cula ing sys em o synch onize he de achmen o M. ma ga i i e a ju enile mussels by
117 exposing ish o high wa e empe a u es o se e al weeks (H uska, 1992). The eby, he
118 empe a u e was daily hea ed 1º up o 17ºC, and he ea e main ained cons an un il he end o
119 he expe imen (± 0.2; Supplemen a y able 1) by employing a empe a u e con ol sys em
120 (Aqua ium Con olle E olu ion, Aqua onica®) and a he mos a (Ako®). Mo eo e , wa e was
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121 p e ea ed by mechanical il a ion and ul a iole i adia ion, and he exchange a e was se a
122 2,000 L/h wi h a deg ee o eci cula ion o 98% (± 0.2). Wa e quali y was pe iodically moni o ed
123 in each ank by measu ing ammonia, ni i e, and ni a e wi h a pho ome e (HI83203, HANNA
124 ins umen s®), and pH and dissol ed oxygen by a pH/dissol ed oxygen me e (PD 650 me e ,
125 Oak on®) (Supplemen a y able 1). Bo h ba ches we e ed wi h a comme cial d y pelle
126 (AquaP o, Sk e ing®) a abou 0.5% body weigh o minimize he inpu o ammonia in o he
127 sys em and he ish mo ali ies we e daily checked.
128 The de ached mussel ju eniles we e daily collec ed om he ank ou le by sie es wi h a mesh
129 size o 150 μm. La e on, hey we e pipe ed, coun ed unde he s e eomic oscope, and classi ied
130 as iable o un iable o cul u ing based on he p esence o absence o pedal and al e
131 mo emen s, espec i ely. Addi ionally, iable ju eniles we e pho og aphed wi h a Leica® M125
132 s e eomic oscope and a M170HD digi al came a. Since he numbe o expe imen al ish was
133 educed due o he sampling p ocedu e, mussel ju eniles coun s we e s anda dized conside ing
134 he numbe o emaining ish le in he ank in ju eniles/ ish.
135 2.4 Sampling p ocedu e: de e mina ion o he ope cula a e and nec opsy
136 Eigh in es ed and con ol ish we e andomly sampled a day 203, 221, 225, 232 and 239 PE,
137 ocusing on he ju enile de achmen . A 246 PE, he emaining ele en ish o each g oup we e
138 sampled o conclude he s udy. A e hand-ne ing, ish we e indi idually held in o indi idual
139 anspa en bucke s wi h 1 L o wa e , and he ope cula mo emen s we e measu ed o 20 s wice
140 (3 and 15 min a e ne cap u e) o calcula e he mean ope cula a e o each ish, exp essed in
141 ope cula bea s/min (OBM).
142 A e wa ds, salmon we e indi idually anes he ized and eu hanized by o e exposu e o a
143 solu ion o 200 mg/L o icaine me hanesul ona e (MS‐222, Sigma‐Ald ich®) bu e ed wi h 400
144 mg/L o sodium bica bona e. Eu hanasia was con i med by sec ioning he spinal co d. Weigh (±
145 0.1 g), o k leng h (± 0.1 cm) and Ful on´s condi ion ac o (100×weigh (g)/leng h (cm)3) we e
146 calcula ed and s anda d de ia ion exp essed (±). Immedia ely, he whole animal was imme sed in
147 wa e in la e al decubi us, he ope culum was abduc ed and he le holob anchs we e
148 s e eomic opho og aphed employing he p e ious equipmen .
149 Subsequen ly, comple e nec opsy o animals was pe o med, and igh holob anchs and o he
150 o gans (skin, hymus, diges i e ac , hea , kidney and spleen) we e sampled and immedia ely
151 ixed in Bouin's ixa i e o 18 h . Fo scanning elec on mic oscopy (SEM), a small po ion o
152 gill issue was ixed i s in 2.5% glu a aldehyde wi h 0.1 M cacodyla e bu e (pH 7.3) and hen
153 in 1% osmium e oxide. Le holob anchs we e dissec ed ou and examined o es ima e he la al
154 load o in es ed ish, s anda dized by ish weigh (la ae/ ish g) acco ding o Ma waha, Aase,
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155 Geis , S oeckle, Kuehn e al. (2019). La e al s e eomic opho og aphs o each le holob anch we e
156 ob ained using he p e ious equipmen o e alua e he la al dis ibu ion in he gill. Addi ional
157 mic opho og aphs we e cap u ed wi h a Leica® DM750 ligh mic oscope a ICC50HD digi al
158 came a o e alua e he encys ed la ae and measu e hei leng h as p e iously desc ibed (n = 80,
159 ± 0.1 μm).
160 All p ocedu es we e ca ied ou a he acili ies o “Cen o Ic iogénico de O Ve al” (Xun a de
161 Galicia) and ollowed he in e na ional (Di ec i e 2010/63/EU, on he p o ec ion o animals used
162 o scien i ic pu poses), na ional (Law 6/2013 and RD 53/2013, on he p o ec ion o animals used
163 o scien i ic expe imen s) and ins i u ional egula ions (USC Re iew Boa d).
164 2.5 Ligh and scanning elec on mic oscopy (SEM)
165 A e Bouin´s ixa ion, igh holob anchs we e decalci ied o 6 h in a 10%
166 e hylenediamine e aace ic acid (EDTA) solu ion (Os eodec, Bio‐op ica®). Decalci ied
167 holob anchs and emaining o gans we e p ocessed o his opa hology by ou ine me hods and
168 sec ions (3 μm) om pa a in‐embedded issue we e s ained wi h hema oxylin and eosin (H&E).
169 Gill issue was addi ionally s ained wi h pe iodic acid-Schi (PAS) and Masson-Goldne
170 ich ome. Slides we e obse ed and pho og aphed using an Olympus® BX51 ligh mic oscopy
171 equipped wi h an DP72 o EP50 digi al came as. On he o he hand, samples o SEM we e
172 dehyd a ed in e hanol solu ions and ou inely p ocessed o examina ion unde a JEOL JSM-
173 6360LV scanning elec on mic oscope.
174 2.6.S a is ical analysis
175 The quan i a i e a iables, body condi ion and ope cula a e, we e s a is ically compa ed
176 be ween g oups employing he Wilcoxon-Mann-Whi ney U es . The s eng h o he ela ionships
177 be ween body condi ion and ope cula a e wi h he pa asi ic loads we e calcula ed du ing he i s
178 h ee samplings (day 203, 221, 225 PE) employing he Spea man´s Co ela ion Coe icien . In
179 addi ion, he daily leng h o he ecen ly de ached ju eniles was included in o he s udy. The
180 signi icance le el was 95% in all cases (p- alue < .05). All he nume ic da a we e analyzed by
181 RS udio so wa e (R Co e Team, 2019).
182 3 RESULTS
183 Du ing he la e glochidiosis, no mo ali ies we e obse ed and ish displayed a mean body
184 condi ion o 0.98 ± 0.08 and a mean ope cula a e a e hand-ne ing o 135 ± 13, wi h simila
185 alues be ween he in es ed and he con ol g oup h oughou mos o he samplings (Figu e S1).
186 Mo eo e , he ju enile de achmen was success ully synch onized be ween day 203 and 238 PE,
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187 yielding a o al o 13,891 ju eniles o M. ma ga i i e a. Du ing his pe iod, he de achmen
188 displayed a le -skewed dis ibu ion wi h a peak o 38 de ached ju eniles/ ish a day 226 PE
189 (Figu e 1a). Based on he cu e o his de achmen , h ee s ages could be es ablished o be e
190 desc ibe he e olu ion o he ju enile de achmen in ela ion wi h he ish pa asi osis and he
191 his opa hological changes (Figu e 1a and b): he ising de achmen s age (day 203–226 PE), he
192 declining de achmen s age (day 227–238 PE) and he pos -de achmen s age (day 239–246 PE).
193 3.1. The ising de achmen s age (day 203–226 PE)
194 A he nec opsy, 96% o ish we e in es ed (Figu e 1b) and ha bo ed a median pa asi ic load
195 o 22.4 ± 47.4 la ae/ ish g, in which highly in es ed ish in he uppe qua ile showed a mean o
196 102 la ae/ ish g; meanwhile, he emaining ish below he uppe qua ile displayed a mean o
197 12.6 la ae/ ish g (Figu e 1c). No signi ican ela ionships we e de ec ed in he co ela ion
198 analysis be ween he pa asi ic load and he body condi ion no he ope cula a e a e hand-
199 ne ing (Figu e 1c).
200 By naked eye isualiza ion o in es ed ish, abundan , well-delimi ed, punc i o m nodules
201 we e obse ed in he b anchial issue (Figu e 2a). By s e eomic oscopy, he imme sed gills
202 e ealed a high numbe o i o y bean-shaped pa asi ic nodules loca ed on he ou holob anchs
203 (Figu e 2b), mos ly a he ailing edges o he ilamen s (Figu e 2c). Fish wi h less han 58
204 la ae/ ish g exhibi ed a dis inc i e dis ibu ion o he la ae, clus e ed in bunches, which ended
205 o ga he a he do sal and en al egions o each holob anch (Figu e 2d).
206 By ligh mic oscopy a low magni ica ions, he pa asi ic clus e s we e composed o se e al
207 la ge and p o uding nodules. Each nodule co esponded wi h a cys o M. ma ga i i e a la a,
208 su ounded by a well-localized epi helial esponse in which adjacen lamellae, and some imes he
209 adjacen ilamen s, we e used obli e a ing he gill exchange su ace (Figu e 2e and ). O en,
210 used lamellae became elonga ed up o wice hei leng h (Figu e 2 and 3c). The pa asi ic cys s
211 we e comple ely co e ed by an in ense epi helial hype plasia and hype ophy, he la e
212 cha ac e ized by he high numbe o epi helial cells wi h la ge swollen cy oplasm, o oid nucleus
213 and small nucleoli (Figu e 2g). Mi o ic igu es we e equen ly de ec ed all o e he cys , al hough
214 hey we e mo e abundan basally a he in e lamella epi helium o he pa asi ized egions (Figu e
215 2 ). The epi helial cells closes o he bi al e pe ios acum concen ically a anged and became
216 hinne , elonga ed and in ensely eosinophilic wi h H&E, being also PAS-posi i e (Figu e 2g).
217 Associa ed o he pa asi osis, a lymphocy ic in lamma o y in il a e was in e spe sed be ween he
218 hype plas ic epi helium (Figu e 2g). Mo eo e , a mixed in lamma o y cell popula ion, composed
219 by mac ophages and polymo phonuclea cells, was iden i ied a he deepe laye s o he ilamen s
220 and ela ed o he in e lamella sys em, pa icula ly unde nea h he basal memb ane o small
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221 ladde -like essels (Figu e 2h) and ma gina ed o e he endo helium o elonga ed sinus (Figu e
222 2i).
223 No o he lesions we e eco ded in o he sampled o gans om in es ed ish. On he o he hand,
224 con ol ish exhibi ed un used lamellae, in aepi helial lymphocy es a he ailing edge o he
225 ilamen s and sca ce in lamma o y cells associa ed o he in e lamella sys em (Figu e 2j).
226 Some pa asi ic nodules bulged o a high ex en on he gill su ace (Figu e 3a), molding he
227 con iguous ilamen s obse ed as ocal a eas wi h conca e shape (Figu e 2e). Fu he mo e, esh
228 mic oscopy highligh ed he undis u bed a e ioles unde nea h he pa asi ic cys (Figu e 3b) and
229 excep ionally de ious a e ioles we e ela ed o he encys ed la ae a he ilamen al ips (Figu e
230 3c). In hese cases, he g ow h plaque o he ilamen was also de ia ed (Figu e 3d) and he
231 ca ilage ay was hickened due o an i egula disposi ion o hype plas ic chond ocy es, obse ed
232 by his ology (Figu e 3e). Unde SEM, he nodula su ace was co e ed by la ened and polygonal
233 epi helial cells, o e laid by well-de ined mic o idges (Figu e 3 ). Howe e , a he mos p o uding
234 egions, a localized epi helial degene a ion occu ed, cha ac e ized by he p esence o ain
235 mic o idges and cell bounda ies (Inse , Figu e 3 ). His ologically, he con ac a ea be ween hese
236 la ge nodules and he ilamen s became educed (50–100 μm wid h) c ea ing a peduncula ed,
237 ea d op shaped s uc u e suppo ed by he hype plas ic epi helium o used lamellae (Figu e 3g).
238 Occasionally, he encys ed pa asi es we e ba ely linked o he gill issue by a e y cons ic ed and
239 pinched-o peduncle which showed a supe icial goble cell hype plasia (Figu e 3h). Mo eo e ,
240 a deepe laye s, he hype plas ic issue was accompanied by mode a e epi helial apop osis,
241 obse ed by pyknosis, cell sh inkage and he p esence o apop o ic bodies (Figu e 3h). Based on
242 he o e all mo phopa hological ea u es desc ibed, la ae sloughed om he gill by a d ople
243 de achmen mechanism, illus a ed in Figu e 3i and simpli ied in h ee sequen ial s eps: 1.
244 p o usion o e he su ounding limi s, 2. pinch-o by educ ion o he con ac a ea be ween he
245 gill and 3. de achmen o he la ae wi hou issue dis up ion.
246 The encys ed la ae displayed a hin and e ingen pe ios acum unde esh mic oscopy,
247 which allowed o disce n he al es comple ely closed, he al e ims acing each o he and he
248 disce nible man le wi hin he in e nal pallial and ex apallial ca i ies (Figu e 3j). Once de ached
249 om he gills, he ecen ly sloughed and iable ju eniles exhibi ed mobile al es and a p o ac ile
250 oo p o uding ou o he al e limi s (Figu e 3k). Taking in o accoun he mo ili y o he al es,
251 he mussel iabili y ab up ly inc eased a day 209 PE onwa ds om alues below 63% o o e
252 93% (Figu e 1a).
253 Adjacen o he pa asi ized a eas, he p oli e a i e lamellae we e a iably used h ough hei
254 ex ension, anging om comple e and solid usions o pa ially used lamellae (Figu e 4a- ). Solid
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465 Cas illo, P. A., Va ela-Dopico, C., Ondina, P., Qui oga, M. I. & Be múdez, R. (2020). Ea ly s ages
466 o Ma ga i i e a ma ga i i e a glochidiosis in A lan ic salmon: Mo phopa hological
467 cha ac e iza ion. Jou nal o Fish Diseases, 43(1), 69-80. doi: 10.1111/j d.13100
468 Chowdhu y, M. M. R., Ma jomäki, T. J. & Taskinen, J. (2019). E ec o glochidia in ec ion on
469 g ow h o ish: eshwa e pea l mussel Ma ga i i e a ma ga i i e a and b own ou
470 Salmo u a. Hyd obiologia. doi: 10.1007/s10750-019-03994-4
471 Colo ni, A. & Bu gess, P. (1997). C yp oca yon i i ans B own 1951, he cause o ‘whi e spo
472 disease’ in ma ine ish: an upda e. Aqua ium Sciences and Conse a ion, 1(4), 217-238.
473 doi: 10.1023/A:1018360323287
474 Cunjak, R. A. & McGladde y, S. E. (1991). The pa asi e–hos ela ionship o glochidia (Mollusca:
475 Ma ga i i e idae) on he gills o young-o - he-yea A lan ic salmon (Salmo sala ).
476 Canadian Jou nal o Zoology, 69(2), 353-358. doi: 10.1139/z91-055
477 Cu elod, A., Seddon, M. & Neube , E. (2011). Eu opean ed lis o non-ma ine molluscs:
478 Publica ions o ice o he Eu opean Union Luxembou g.
479 Dang, M., Pi man, K., Sonne, C., Hansson, S., Bach, L., Sønde gaa d, J., S ide, M. & Nowak, B.
480 (2020). His ological mucous cell quan i ica ion and mucosal mapping e eal di e en
481 aspec s o mucous cell esponses in gills and skin o sho ho n sculpins (Myoxocephalus
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484 ou , Salmo gai dne i Richa dson. Jou nal o Fish Diseases, 9(4), 313-318. doi:
485 10.1111/j.1365-2761.1986. b01020.x
486 Denic, M., Taeube , J. E. & Geis , J. (2015). T ophic ela ionships be ween he la ae o wo
487 eshwa e mussels and hei ish hos s. In e eb a e Biology, 134(2), 129-135. doi:
488 10.1111/i b.12080
489 Ewing, M. S. & Kocan, K. M. (1987). Ich hyoph hi ius mul i iliis (Ciliopho a) Exi om Gill
490 Epi helium. Jou nal o P o ozoology, 34(3), 309-312. doi: 10.1111/j.1550-
491 7408.1987. b03181.x
492 Filipsson, K., B ijs, J., Näslund, J., Wengs öm, N., Adamsson, M., Zá o ka, L., Ös e ling, E. M. &
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494 ma ga i i e a) la ae coincides wi h inc eased me abolic a e and haema oc i in
495 ju enile b own ou (Salmo u a). Pa asi ology Resea ch, 116(4), 1353–1360. doi:
496 10.1007/s00436-017-5413-2
497 Geis , J. (2010). S a egies o he conse a ion o endange ed eshwa e pea l mussels
498 (Ma ga i i e a ma ga i i e a, L.): a syn hesis o Conse a ion Gene ics and Ecology.
499 Hyd obiologia, 644(1), 69-88. doi: 10.1007/s10750-010-0190-2
500 Gilmou , K. M. & Pe y, S. F. (2018). Con lic and Comp omise: Using Re e sible Remodeling o
501 Manage Compe ing Physiological Demands a he Fish Gill. Physiology, 33(6), 412-422.
502 doi: 10.1152/physiol.00031.2018
503 G izzle, J. M. & Ki yu, Y. (1993). His opa hology o Gill, Li e , and Panc eas, and Se um Enzyme
504 Le els o Channel Ca ish In ec ed wi h Ae omonas hyd ophila Complex. Jou nal o
505 Aqua ic Animal Heal h, 5(1), 36-50. doi: 10.1577/1548-
506 8667(1993)005<0036:HOGLAP>2.3.CO;2
507 Gum, B., Lange, M. & Geis , J. (2011). A c i ical e lec ion on he success o ea ing and cul u ing
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514 Hende son, D. M., P i cha d, W. G. & Smolka, L. B. (1997). On he pinch-o o a pendan d op o
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525 Kaise , B. E. (2005). The e ec s o glochidiosis on ish espi a ion. (Doc o al disse a ion, Missou i
526 S a e Uni e si y, Sp ing ield, Missou i). Re ie ed om
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528 Ka na, D. W. & Millemann, R. E. (1978). Glochidiosis o salmonid ishes. III. Compa a i e
529 suscep ibili y o na u al in ec ion wi h Ma ga i i e a ma ga i i e a (L.) (Pelecypoda:
530 Ma ga i anidae) and associa ed his opa hology. Jou nal o Pa asi ology, 64(3), 528-537.
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532 Kudo, S. & Kimu a, N. (1983). The Reco e y om Hype plasia in an A i icial In ec ion. Bulle in o
533 he Japanese Socie y o Scien i ic Fishe ies, 49(11), 1635-1641. doi:
534 10.2331/suisan.49.1635
535 Kuma , R., Madha i, R. & Sailaja, B. (2017). Spa ial dis ibu ion o ec opa asi es on he gills o
536 he mulle , Liza mac olepis: he e ec s o pollu ion. Jou nal o Pa asi ic Diseases, 41(1),
537 40-47. doi: 10.1007/s12639-015-0746-1
538 Lois, S., Ondina, P., Ou ei o, A., Ama o, R. & San Miguel, E. (2014). The no h-wes o he Ibe ian
539 Peninsula is c ucial o conse a ion o Ma ga i i e a ma ga i i e a (L.) in Eu ope.
540 Aqua ic Conse a ion: Ma ine and F eshwa e Ecosys ems, 24(1), 35-47. doi:
541 10.1002/aqc.2352
542 Ma waha, J., Aase, H., Geis , J., S oeckle, B. C., Kuehn, R. & Jakobsen, P. J. (2019). Hos (Salmo
543 u a) age in luences esis ance o in es a ion by eshwa e pea l mussel
544 (Ma ga i i e a ma ga i i e a) glochidia. Pa asi ology Resea ch, 118(5), 1519-1532. doi:
545 10.1007/s00436-019-06300-2
546 Medzhi o , R., Schneide , D. S. & Soa es, M. P. (2012). Disease Tole ance as a De ense S a egy.
547 Science, 335(6071), 936-941. doi: 10.1126/science.1214935
548 Muelle , M. E., Sanchez, D. A., Be gman, H. L., McDonald, D. G., Rhem, R. G. & Wood, C. M.
549 (1991). Na u e and Time Cou se o Acclima ion o Aluminum in Ju enile B ook T ou
550 (Sal elinus on inalis). II. Gill His ology. Canadian Jou nal o Fishe ies and Aqua ic
551 Sciences, 48(10), 2016-2027. doi: 10.1139/ 91-240
552 Nilsson, G. E. (2007). Gill emodeling in ish–a new ashion o an ancien sec e ? Jou nal o
553 Expe imen al Biology, 210(14), 2403-2409. doi: 10.1242/jeb.000281
554 Olson, K. R. (2002). Vascula ana omy o he ish gill. Jou nal o Expe imen al Zoology, 293(3),
555 214-231. doi: 10.1002/jez.10131
556 Ooue, K., Te ui, A., U abe, H. & Nakamu a, F. (2017). A delayed e ec o he aqua ic pa asi e
557 Ma ga i i e a lae is on he g ow h o he salmonid hos ish Onco hynchus masou
558 masou. Limnology, 18(3), 345-351. doi: 10.1007/s10201-017-0514-2
559 Ös e ling, M. E. (2011). Tes and applica ion o a non-des uc i e pho o-me hod in es iga ing
560 he pa asi ic s age o he h ea ened mussel Ma ga i i e a ma ga i i e a on i s hos ish
561 Salmo u a. Biological Conse a ion, 144(12), 2984-2990. doi:
562 10.1016/j.biocon.2011.09.001
563 Ös e ling, M. E., Fe m, J. & Piccolo, J. J. (2014). Pa asi ic eshwa e pea l mussel la ae
564 (Ma ga i i e a ma ga i i e a L.) educe he d i - eeding a e o ju enile b own ou
565 (Salmo u a L.). En i onmen al Biology o Fishes, 97(5), 543-549. doi: 10.1007/s10641-
566 014-0251-x
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567 Powell, M. D., Lee , M. J., Robe s, S. D. & Jones, M. A. (2008). Neopa amoebic gill in ec ions:
568 Hos esponse and physiology in salmonids. Jou nal o Fish Biology, 73(9), 2161-2183.
569 doi: 10.1111/j.1095-8649.2008.02053.x
570 P es on, S. J., Keys, A. & Robe s, D. (2007). Cul u ing eshwa e pea l mussel Ma ga i i e a
571 ma ga i i e a: a b eak h ough in he conse a ion o an endange ed species. Aqua ic
572 Conse a ion: Ma ine and F eshwa e Ecosys ems, 17(5), 539-549. doi: 10.1002/aqc.799
573 R Co e Team. (2019). R: a language and en i onmen o s a is ical compu ing: R Founda ion o
574 S a is ical Compu ing, Vienna, Aus ia. Re ie ed om h ps://www.R-p ojec .o g/
575 Ronza, P., Villama ín, A., Méndez, L., Pa do, B. G., Be múdez, R. & Qui oga, M. I. (2019).
576 Immunohis ochemical exp ession o E–cadhe in in di e en issues o he eleos ish
577 Scoph halmus maximus. Aquacul u e, 501, 465-472. doi:
578 10.1016/j.aquacul u e.2018.12.009
579 Rumme , J. L., Wang, S., S e ensen, J. F. & Randall, D. J. (2014). Func ion and con ol o he ish
580 seconda y ascula sys em, a con as o mammalian lympha ic sys ems. The Jou nal o
581 expe imen al biology, 217(5), 751-757. doi: 10.1242/jeb.086348
582 Sales, C. F., San os, K. P. E. d., Rizzo, E., Ribei o, R. I. M. d. A., San os, H. B. d. & Thomé, R. G.
583 (2017). P oli e a ion, su i al and cell dea h in ish gills emodeling: F om inju y o
584 eco e y. Fish & Shell ish Immunology, 68, 10-18. doi: 10.1016/j. si.2017.07.001
585 Scha sack, G. (1994). Lich - und elek onenmik oskopische Un e suchungen an La als adien
586 einheimische Unionacea (Bi al ia; Eulamellib anchia a) [Ligh and elec on mic oscopic
587 s udies on la al s ages o na i e Unionacea (Bi al ia, Eulamellib anchia a)].
588 (Unpublished doc o al disse a ion), Uni e si ä Hanno e , Hanno e , Ge many.
589 Re ie ed om h p://www. epo.uni-hanno e .de/handle/123456789/5643
590 Schmid , J. G. (2013). Wound healing in ainbow ou (Onco hynchus mykiss) and common ca p
591 (Cyp inus ca pio) wi h a ocus on gene exp ession and wound imaging. Technical
592 Uni e si y o Denma k.
593 Sollid, J., De Angelis, P., Gunde sen, K. & Nilsson, G. E. (2003). Hypoxia induces adap i e and
594 e e sible g oss mo phological changes in c ucian ca p gills. Jou nal o Expe imen al
595 Biology, 206, 3667-3673. doi: 10.1242/jeb.00594
596 Spea e, D. J., Ca ajal, V. & Ho ney, B. S. (1999). G ow h Supp ession and B anchi is in T ou
597 Exposed o Hyd ogen Pe oxide. Jou nal o Compa a i e Pa hology, 120(4), 391-402. doi:
598 10.1053/jcpa.1998.0285
599 Spea e, D. J., Fe guson, H. W., Beamish, F. W. M., Yage , J. A. & Yamashi o, S. (1991). Pa hology
600 o bac e ial gill disease: ul as uc u e o b anchial lesions. Jou nal o Fish Diseases,
601 14(1), 1-20. doi: 10.1111/j.1365-2761.1991. b00572.x
602 S o he , J. A. (2013). Hyd odynamic esis ance and low pa e ns in he gills o a ilapine ish.
603 The Jou nal o expe imen al biology, 216(14), 2595. doi: 10.1242/jeb.079517
604 S een, L., Ka lsen, C. & Y ebo g, E. (2020). Mechanical induced wounds in ish – a e iew on
605 models and healing mechanisms. Re iews in Aquacul u e, n/a(n/a). doi:
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608 s ains as hos s o he endange ed eshwa e pea l mussel (Ma ga i i e a
609 ma ga i i e a L.). Aqua ic Conse a ion: Ma ine and F eshwa e Ecosys ems, 20(7), 728-
610 734. doi: 10.1002/aqc.1147
611 Taeube , J. E. & Geis , J. (2013). C i ical swimming speed o b own ou (Salmo u a) in es ed
612 wi h eshwa e pea l mussel (Ma ga i i e a ma ga i i e a) glochidia and implica ions
613 o a i icial b eeding o an endange ed mussel species. Pa asi ology Resea ch, 112(4),
614 1607-1613. doi: 10.1007/s00436-013-3314-6
615 Taeube , J. E., Gum, B. & Geis , J. (2013). Va iable de elopmen and excys men o eshwa e
616 pea l mussel (Ma ga i i e a ma ga i i e a L.) a cons an empe a u e. Limnologica -
617 Ecology and Managemen o Inland Wa e s, 43(4), 319-322. doi:
618 10.1016/j.limno.2013.01.002
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619 Takeichi, M. (2014). Dynamic con ac s: ea anging adhe ens junc ions o d i e epi helial
620 emodelling. Na u e Re iews Molecula Cell Biology, 15(6), 397-410. doi:
621 10.1038/n m3802
622 Thomas, G. R., Taylo , J. & Ga cia de Leaniz, C. (2013). Does he pa asi ic eshwa e pea l mussel
623 M. ma ga i i e a ha m i s hos ? Hyd obiologia, 735(1), 191-201. doi: 10.1007/s10750-
624 013-1515-8
625 T easu e , J. W., Has ie, L. C., Hun e , D., Duncan, F. & T easu e , C. M. (2006). E ec s o
626 (Ma ga i i e a ma ga i i e a) glochidial in ec ion on pe o mance o ank- ea ed
627 A lan ic salmon (Salmo sala ). Aquacul u e, 256(1–4), 74-79. doi:
628 10.1016/j.aquacul u e.2006.02.031
629 T easu e , J. W. & Tu nbull, T. (2000). The pa hology and seawa e pe o mance o a med
630 A lan ic salmon in ec ed wi h glochidia o Ma ga i i e a ma ga i i e a. Jou nal o Fish
631 Biology, 57(4), 858-866. doi: 10.1111/j.1095-8649.2000. b02197.x
632 Tubbs, L., Wybou ne, B. A. & Lumsden, J. S. (2010). Nodula gill disease causing p oli e a i e
633 b anchi is and mo ali y in Chinook salmon (Onco hynchus shawy scha). N Z Ve J,
634 58(1), 59-61. doi: 10.1080/00480169.2010.65061
635 Tumbol, R. A., Powell, M. D. & Nowak, B. F. (2001). Ionic E ec s o In ec ion o Ich hyoph hi ius
636 mul i iliis in Gold ish. Jou nal o Aqua ic Animal Heal h, 13(1), 20-26. doi: 10.1577/1548-
637 8667(2001)013<0020:IEOIOI>2.0.CO;2
638 Vaughn, C. C. (2017). Ecosys em se ices p o ided by eshwa e mussels. Hyd obiologia, 810(1),
639 15-27. doi: 10.1007/s10750-017-3139-x
640 Wäch le , K., D ehe -Mansu , M. & Rich e , T. (2001). La al Types and Ea ly Pos la al Biology
641 in Naiads (Unionoida). In G. Baue & K. Wäch le (Eds.), Ecology and E olu ion o he
642 F eshwa e Mussels Unionoida (Vol. 145, pp. 93-125): Sp inge Be lin Heidelbe g.
643 Wacke , S., La sen, B. M., Ka lsson, S. & Hinda , K. (2019). Hos speci ici y d i es gene ic
644 s uc u e in a eshwa e mussel. Scien i ic Repo s, 9(1), 10409. doi: 10.1038/s41598-
645 019-46802-8
646 Walle , D. L. & Mi chell, L. G. (1989). Gill issue eac ions in walleye S izos edion i eum i eum
647 and common ca p Cyp inus ca pio o glochidia o he eshwa e mussel Lampsilis
648 adia a siliquoidea. Diseases o Aqua ic O ganisms, 6(2), 81-87. doi: 10.3354/dao006081
649 Woo en, R. (1974). The spa ial dis ibu ion o Dac ylogy us amphibo h ium on he gills o u e
650 Gymnocephalus ce nua and i s ela ion o he ela i e amoun s o wa e passing o e
651 he pa s o he gills. Jou nal o Helmin hology, 48(03), 167-174. doi:
652 10.1017/S0022149X00022793
653
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654 Figu es
655 Figu e 1. (a and b) E olu ion o he mussel ju enile de achmen (a) in ela ion wi h he ish
656 pa asi osis (b) du ing he h ee s ages o la e glochidiosis o M. ma ga i i e a delimi ed by dashed
657 ed lines. (c) No co ela ion be ween he pa asi ic loads and he ish body condi ion no he
658 ope cula a e we e de ec ed in he co ela ion analysis. Indi iduals wi h pa asi ic loads in he
659 uppe qua ile we e ep esen ed in ed. Con idence in e al was 95%.
660 Figu e 2. Main mo phopa hological indings du ing he ising de achmen s age o la e M.
661 ma ga i i e a glochidiosis. (a) Gill mac opho og aphy o an in es ed ish wi h punc i o m lesions
662 which co esponded wi h he encys ed la ae (a ows). (b and c) S e eomic opho og aphs
663 showing he bean-shaped la ae loca ed be ween each pai o hemib anchs indica ed wi h
664 a owheads. (d) Dis ibu ion o pa asi es clus e ed in bunches a he do sal and en al egions o
665 each holob anch (as e isks). (e) The localized epi helial esponse enclosing he pa asi ic cys s was
666 accompanied by se e e lamella and ilamen al usions (as e isks). No e he conca e dep essions
667 on he ilamen su ace con iguous o an adjacen la a (a ows). H&E s ain. ( ) Su ound he
668 ou e su ace o he pa asi ic cys (a owheads), he used and elonga ed lamellae (dashed lines)
669 we e ela ed o abundan mi o ic igu es (ci cles) and in e lamella cys s (a ow). No e he
670 pleomo phic in lamma o y in il a e loca ed a he in e lamella sys em (as e isks). H&E s ain.
671 (g) A high numbe o lymphocy es in e spe sed be ween epi helial cells, he la e became
672 la ened and eosinophilic owa ds he la ae (a owheads). H&E s ain. Inse : The eosinophilic
673 epi helial cells (a ows) beside he pa asi ic cys s (a owheads) became hinne and mo e in ensely
674 s ained unde he PAS s ain. (h) In e lamella sys em wi h a pleomo phic in lamma o y (as e isks)
675 unde nea h he in ica e basemen memb ane highligh ed wi h he Masson-Goldne ich ome
676 s ain. (i and j) Compa ison be ween in es ed ish wi h ma gina ed mac ophages and
677 polymo phonuclea cells o e he in e lamella sys em endo helia (a owheads, i) and con ol ish
678 showing a no mal lamella mo phology wi hou leukocy es o e he endo helial cells lining he
679 in e lamella sys em (a owheads, j). H&E s ains.
680 Figu e 3. Gill mo phopa hology o he mos supe icial pa asi ic cys s du ing he ising
681 de achmen s age o la e M. ma ga i i e a glochidiosis (a-h) and he d ople de achmen (i) om
682 encys ed la ae in o ee-li ing ju eniles (j and k). (a) Scanning elec on mic oscopy (SEM) o a
683 clus e o buoyan encys ed la ae (as e isks). (b and c) F esh mic opho og aphs compa ing he
684 unal e ed a e ioles (b, a owheads) wi h excep ionally de ious a e ioles (c, a owheads). (d and
685 e) De ia ion o he g ow h plaque (a owheads, d) and a well-localized ca ilage hype plasia
686 (a owheads, e) o e g owing o e he ca ilage ay (as e isk, e). H&E and Masson-Goldne
687 ich ome s ain, espec i ely. ( ) SEM o a pa asi ic cys comple ely co e ed by epi helial cells
688 wi h he dashed a ea highligh ing he mos p o uding a ea. Inse : De ail o he dashed a ea wi h
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689 poo ly disce nible mic o idges o e he epi helial su ace (a owheads). (g) His ological
690 mic opho og aph o he hype plas ic peduncles (as e isk) ancho ing he la ae o he gill
691 ilamen s. No e he p esence o an elonga e lamella (dashed line). H&E s ain. (h) Cons ic ed
692 peduncle wi h a high numbe o goble cells lined up he epi helial su ace (as e isks) and high
693 numbe o apop o ic (a owheads) and phagocy ic bodies (a ows) a deepe egions. PAS s ain.
694 (i) Schema ic illus a ion o he h ee-s eps d ople de achmen o he mussel ju eniles. (j) F esh
695 ligh mic oscopy o an encys ed la ae wi h he al e ims acing each o he (a owheads) and
696 hei in e nal pallial ca i y (as e isk). (k) S e eomic opho og aph o ecen ly de ached and iable
697 ju eniles wi h hei p o ac ile oo (a owheads).
698 Figu e 4. Fusions and in e lamella ca i ies adjacen o he encys ed la ae du ing he ising
699 de achmen s age o la e M. ma ga i i e a glochidiosis. (a) The limi s o he in e lamella ca i ies
700 we e ba ely isible by esh mic oscopy (a owheads) close o he pa asi ic cys s (as e isks). Inse .
701 De ail o an in e lamella cys associa ed wi h wo used lamellae (a owheads). (b) Solid lamella
702 usion (dashed lines) associa ed wi h he p esence o abundan in e lamella mi o ic igu es
703 (enci cled) and goble cells a he su ace (as e isks). PAS s ain. (c and d). Pa ially used lamellae
704 cha ac e ized by he p esence o in e lamella cys s (as e isks) and cle s (a owheads) su ounded
705 by mucous cells. PAS s ains. (e) SEM o wo used ilamen s (dashed line) wi h small openings
706 loca ed be ween pa ially used lamellae (a owheads). ( ) De ail o he dashed a ea highligh ing
707 wo in e lamella openings (as e isks) lined up by epi helial cells.
708 Figu e 5. Gill mo phopa hology du ing he declining de achmen s age (a–h) and he pos -
709 de achmen s age o la e M. ma ga i i e a glochidiosis (i–k). (a) S e eomic oscopical image o
710 diso de ed lamellae obse ed by ousled seconda y ilamen s (a owheads). (b) By his ology,
711 al e ed lamellae anged om hype plas ic and used lamellae (as e isks) o elonga ed and ben
712 lamellae (a owheads). H&E s ain. (c) De ail o elonga ed lamellae wi h hei ips ben inwa ds
713 and sligh ly hype plas ic (a owheads). No e he p esence o a mode a e numbe o pleomo phic
714 in lamma o y cells a he in e lamella sys em (as e isks). H&E s ain. (d) Hype plas ic and used
715 lamella ips (a owheads) gi ing ise o an elonga ed in e lamella ca i y (as e isk). H&E s ain.
716 (e) De ail o hype plas ic lamellae wi h mos o he goble cells a anged owa ds he in e lamella
717 cle s (a owheads) wi h openings owa ds he su ace (a owheads). PAS s ain. ( ) Pa ially used
718 lamellae showed a eas o mode a e lymphocy ic (as e isks) and localized a eas o epi helial
719 degene a ion (a ows). H&E s ain. (g) De ailed mic opho og aph o abundan apop o ic bodies
720 (as e isks) and acuolized, swollen and sloughing epi helial cells a he ou e su ace
721 (a owheads). H&E s ain. (h) Phagocy osis o apop o ic bodies (a owheads) and small PAS-
722 posi i e acuoles (a ows) was associa ed wi h an a ea o lamella usion. PAS s ain. (i and j)
723 No mal s uc u e o ou e su ace o he gill by s e eomic oscopy (i) and scanning elec on
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724 mic oscopy (j). (k) His ological pho og aph showing mild epi helial hype plasia, elonga ions and
725 lamella de ia ion (a owheads) oge he wi h a mode a e numbe o mononuclea cells a he
726 in e lamella sys em (as e isks). H&E s ain.
727 Supplemen a y able and igu e a e included in Suppo ing In o ma ion.
728
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