Morphopatology and gill recovery of Atlantic salmon during the parasitic detachment of Margaritifera margaritifera

Review Copy Morphopatology and gill recovery of Atlantic salmon during the parasitic detachment of Margaritifera margaritifera Journal: Journal of Fish Diseases Manuscript ID JFD-2021-44 Wiley - Manuscript type: Research Article Date Submitted by the Author: 03-Feb-2021 Complete List of Authors: Castrillo, Pedro A.; Universidade de Santiago de Compostela Varela-Dopico, Catuxa; Universidade de Santiago de Compostela Bermúdez, Roberto; Universidade de Santiago de Compostela Ondina, Paz; Universidade de Santiago de Compostela Quiroga, Mª Isabel; Universidade de Santiago de Compostela Keywords: Keywords: gill healing, parasite detachment, <i>Salmo salar</i>, glochidiosis, Freshwater Pearl Mussel Journal of Fish Diseases Journal of Fish Diseases Review Copy 1 1Morphopatology and gill recovery of Atlantic salmon during the parasitic detachment of 2Margaritifera margaritifera 3Short running title: Gill recovery after the parasite detachment 4Castrillo, Pedro A.1; Varela-Dopico, Catuxa2; Bermúdez, Roberto1,3; Ondina, Paz2; Quiroga, 5María Isabel1,3 61. Department of Anatomy, Animal Production and Veterinary Clinical Sciences, Faculty of 7Veterinary, Universidade de Santiago de Compostela, Lugo, Spain. 82. Department of Zoology, Genetics and Physical Anthropology, Faculty of Veterinary, 9Universidade de Santiago de Compostela, Lugo, Spain. 10 3. Instituto de Acuicultura, Universidade de Santiago de Compostela, 15705, Santiago de 11 Compostela, Spain. 12 ORCID: Pedro A. Castrillo: 0000-0001-5499-7190; Catuxa Varela-Dopico: 0000-0002- 13 3513-1444; Roberto Bermúdez Pose: 0000-0003-4969-4122; Paz Ondina: 0000-0003-4392-9250; 14 María Isabel Quiroga: 0000-0001-6832-7665 15 Correspondence 16 Roberto Bermúdez, Department of Anatomy, Animal Production and Veterinary Clinical 17 Sciences, Faculty of Veterinary, Universidade de Santiago de Compostela, Campus Universitario 18 s/n, 27002, Lugo, Spain. Email: [email protected] 19 Acknowledgments 20 The authors would like to thank the assistance of Dr. R. Mascato and R. Ocharan with the 21 field work and the captivity procedures during this long-term experimental trial. Also, we warmly 22 thanks S. Maceiras for the thorough histopathological technical assistance and Dr. A. M. de 23 Azevedo, Dr. A. P. Losada and Dr. P. Ronza for the scientific advices. The conservation 24 programme was cofunded by the “Fundación Biodiversidad” within the MarMaCul and 25 MargaSalmo Projects and by the Xunta de Galicia within “Programa de Consolidación e 26 Estructuración de Unidades de Investigación Competitivas” (ED4313 2019/24 and ED431D 27 2017/22) for the regional development for scientific network. P. A. Castrillo held a University 28 Professorship Formation (FPU) grant from the Spanish Ministry of Education, Culture and Sport 29 (FPU17/02004). We also acknowledge the support of the research collaboration agreement with 30 the Consellería de Medio Ambiente, Territorio y Vivienda (Xunta de Galicia). Page 1 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 31 Data availability statement 32 The data that support the findings of this study are available from the corresponding author 33 upon reasonable request. 34 Conflict of interest statement 35 The authors declare that they have no competing interests. 36 Abstract 37 During the conservation aquaculture of the freshwater mussel Margaritifera margaritifera, 38 fish health has become a concern due the need of mussel larvae (glochidia) to parasitize the 39 salmonid gills and metamorphose into juveniles. However, there is lack of information about the 40 impact on fish during the juvenile detachment and the subsequent gill healing. To evaluate the 41 morphopathological changes and gill recovery after the parasitism of M. margaritifera, fifty-one 42 Atlantic salmon fry (Salmo salar), infested with around 22 larvae/fish g, were necropsied during 43 the synchronized detachment of the mussel juveniles, and gills were assessed by 44 stereomicroscopy, and by light and scanning electron microscopy. Salmon showed no clinical 45 signs during the trial and gills recovered their normal morphology almost completely in a short 46 time, suggesting a minimal impact on fish health after glochidiosis. In this sense, the non-erosive 47 droplet detachment and the goblet cell hyperplasia favored an effective gill remodeling mediated 48 by apoptosis, polarization and cell shedding of the gill epithelia, providing insights to the defense, 49 clearing and healing mechanisms of the gill. These morphopathological techniques could also be 50 implemented to preserve fish welfare and to optimize the artificial breeding programmes of 51 endangered freshwater mussels. 52 Keywords 53 gill healing, Freshwater Pearl Mussel, fish pathology, parasite detachment, Salmo salar, 54 glochidiosis Page 2 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 3 55 1 Introduction 56 Freshwater mussels are bivalves with extraordinary capacities of biofiltration and burrowing, 57 thus, they provide significant ecosystem services and also indirectly protect many other species 58 (Vaughn, 2017). Despite its importance, certain naiads as Margaritifera margaritifera (L., 1758) 59 are categorized in the European Red List of IUCN as Critically Endangered due to the serious 60 decline of the populations (Cuttelod, Seddon & Neubert, 2011). To protect the most affected 61 populations, which lack natural recruitment (Lois, Ondina, Outeiro, Amaro & San Miguel, 2014), 62 one emergency conservation strategy is to implement artificial breeding programmes (Gum, 63 Lange & Geist, 2011). 64 The culturing techniques of freshwater mussels depend on the compulsory larval parasitic 65 stage on Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.) fry, regarded as a 66 mechanism of dispersal, nutrition and protection (Barnhart, Haag & Roston, 2008; Denic, 67 Taeubert & Geist, 2015; Geist, 2010). In particular, the larva of M. margaritifera clamps and 68 encysts into the salmonid gills for several months until it detaches in spring––once temperature 69 rises over 15 ºC––as a free-living juvenile (Hruska, 1992; Taeubert, Gum & Geist, 2013). 70 Accordingly, fish suffer a multifocal proliferative branchitis, which leads to a disease status 71 known as glochidiosis, given by the name of the infesting larvae, the glochidium (Karna & 72 Millemann, 1978). 73 This host-parasite interaction supposes a bottleneck for freshwater mussel aquaculture and a 74 concern towards the welfare of the host fish. In an attempt to optimize the culturing efforts several 75 studies had established certain recommended glochidial loads (Taeubert & Geist, 2013), based on 76 the impact on fish survival, growth, respiration, metabolism, swimming capacity and fish 77 behavior (Chowdhury, Marjomäki & Taskinen, 2019; Cunjak & McGladdery, 1991; Filipsson, 78 Brijs, Näslund, Wengström, Adamsson et al., 2017; Österling, Ferm & Piccolo, 2014; Preston, 79 Keys & Roberts, 2007; Thomas, Taylor & Garcia de Leaniz, 2013). However, there is a lack of 80 data about the pathogenesis of the disease, in which the morphopathological evaluation of the gill 81 lesions remain overlooked, mostly focused on the premature rejection of unviable larvae, which 82 is associated to an erosive branchitis during early stages of glochidiosis (Castrillo, Varela-Dopico, 83 Ondina, Quiroga & Bermúdez, 2020). This contrast with the extensive knowledge of other gill 84 ectoparasites which compromise the fish welfare in aquaculture, e.g., Neoparamoeba perurans 85 and Ichthyophthirius multifiliis (Powell, Leef, Roberts & Jones, 2008; Tumbol, Powell & Nowak, 86 2001). Lastly, gills are able to recover after removal of the inciting cause as chemicals or 87 infectious agents (Daoust & Ferguson, 1986; Kudo & Kimura, 1983; Sales, Santos, Rizzo, 88 Ribeiro, Santos et al., 2017; Speare, Carvajal & Horney, 1999); nevertheless, very few studies Page 3 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 89 refer to the recovery of lesions after glochidiosis (Kaiser, 2005; Karna & Millemann, 1978; 90 Scharsack, 1994; Treasurer & Turnbull, 2000). 91 Employing this artificial glochidiosis as a model of parasitic gill disease and recovery in 92 Atlantic salmon, the purpose of this study was to perform a comprehensive morphopathological 93 evaluation during the late stages of glochidiosis including the juvenile detachment and the 94 subsequent gill healing process. Hence, this study may also provide insights to understand this 95 complex host-parasite interaction and to preserve fish welfare during the rearing of these 96 important and endangered freshwater mussels. 97 2 MATERIAL AND METHODS 98 2.1 Experimental gill infestation and selection of infested fish 99 Experimental fish came from an artificial glochidiosis trial performed in September 2015 100 (Castrillo et al., 2020). Briefly, 1000 salmon fry were exposed by bath immersion to M. 101 margaritifera glochidia (1,000 glochidia /fish gram) collected from gravid wild mussels. A group 102 of non-exposed fish served as control group. At day 14 post-exposure (PE) an early detachment 103 of inviable glochidia occurred and successfully infected fish could be only identified by means of 104 light microscopy, due to the larval size at this stage (diameter of 70 μm). 105 After this early rejection, the remaining salmon were reared for six more months to allow the 106 development of the encysted larvae and became macroscopically visible on the gill (diameter of 107 350 μm). Thus, after 202 days postexposure (PE), each exposed fish could be in vivo diagnosed 108 as infested or non-infested by manual immobilization, abduction of the opercula and gill 109 visualization. This diagnostic procedure was performed in less than four seconds. As a result, 51 110 out of 600 exposed fish were selected as infested (prevalence of 8.5%). The same exploratory 111 procedure was performed on 51 non-exposed fish. Moreover, to ensure an optimal fish health 112 status and confirm the absence of any other pathological processes prior the in vivo procedure, 113 five exposed and non-exposed fish were necropsied and processed for histopathology. 114 2.3 Synchronization of juvenile detachment 115 Infested and control fish (n = 51, mean weight 5.8 g and length 8.1 cm) were relocated into a 116 recirculating system to synchronize the detachment of M. margaritifera juvenile mussels by 117 exposing fish to high water temperatures for several weeks (Hruska, 1992). Thereby, the 118 temperature was daily heated 1º up to 17ºC, and thereafter maintained constant until the end of 119 the experiment (± 0.2; Supplementary table 1) by employing a temperature control system 120 (Aquarium Controller Evolution, Aquatronica®) and a thermostat (Ako®). Moreover, water was Page 4 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 5 121 pretreated by mechanical filtration and ultraviolet irradiation, and the exchange rate was set at 122 2,000 L/h with a degree of recirculation of 98% (± 0.2). Water quality was periodically monitored 123 in each tank by measuring ammonia, nitrite, and nitrate with a photometer (HI83203, HANNA 124 instruments®), and pH and dissolved oxygen by a pH/dissolved oxygen meter (PD 650 meter, 125 Oakton®) (Supplementary table 1). Both batches were fed with a commercial dry pellet 126 (AquaPro, Skretting®) at about 0.5% body weight to minimize the input of ammonia into the 127 system and the fish mortalities were daily checked. 128 The detached mussel juveniles were daily collected from the tank outlet by sieves with a mesh 129 size of 150 μm. Later on, they were pipetted, counted under the stereomicroscope, and classified 130 as viable or unviable for culturing based on the presence or absence of pedal and valve 131 movements, respectively. Additionally, viable juveniles were photographed with a Leica® M125 132 stereomicroscope and a M170HD digital camera. Since the number of experimental fish was 133 reduced due to the sampling procedure, mussel juveniles counts were standardized considering 134 the number of remaining fish left in the tank in juveniles/fish. 135 2.4 Sampling procedure: determination of the opercular rate and necropsy 136 Eight infested and control fish were randomly sampled at day 203, 221, 225, 232 and 239 PE, 137 focusing on the juvenile detachment. At 246 PE, the remaining eleven fish of each group were 138 sampled to conclude the study. After hand-netting, fish were individually held into individual 139 transparent buckets with 1 L of water, and the opercular movements were measured for 20 s twice 140 (3 and 15 min after net capture) to calculate the mean opercular rate of each fish, expressed in 141 opercular beats/min (OBM). 142 Afterwards, salmon were individually anesthetized and euthanized by overexposure to a 143 solution of 200 mg/L of tricaine methanesulfonate (MS‐222, Sigma‐Aldrich®) buffered with 400 144 mg/L of sodium bicarbonate. Euthanasia was confirmed by sectioning the spinal cord. Weight (± 145 0.1 g), fork length (± 0.1 cm) and Fulton´s condition factor (100×weight (g)/length (cm)3) were 146 calculated and standard deviation expressed (±). Immediately, the whole animal was immersed in 147 water in lateral decubitus, the operculum was abducted and the left holobranchs were 148 stereomicrophotographed employing the previous equipment. 149 Subsequently, complete necropsy of animals was performed, and right holobranchs and other 150 organs (skin, thymus, digestive tract, heart, kidney and spleen) were sampled and immediately 151 fixed in Bouin's fixative for 18 hr. For scanning electron microscopy (SEM), a small portion of 152 gill tissue was fixed first in 2.5% glutaraldehyde with 0.1 M cacodylate buffer (pH 7.3) and then 153 in 1% osmium tetroxide. Left holobranchs were dissected out and examined to estimate the larval 154 load of infested fish, standardized by fish weight (larvae/fish g) according to Marwaha, Aase, Page 5 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 155 Geist, Stoeckle, Kuehn et al. (2019). Lateral stereomicrophotographs of each left holobranch were 156 obtained using the previous equipment to evaluate the larval distribution in the gill. Additional 157 microphotographs were captured with a Leica® DM750 light microscope a ICC50HD digital 158 camera to evaluate the encysted larvae and measure their length as previously described (n = 80, 159 ± 0.1 μm). 160 All procedures were carried out at the facilities of “Centro Ictiogénico de O Veral” (Xunta de 161 Galicia) and followed the international (Directive 2010/63/EU, on the protection of animals used 162 for scientific purposes), national (Law 6/2013 and RD 53/2013, on the protection of animals used 163 for scientific experiments) and institutional regulations (USC Review Board). 164 2.5 Light and scanning electron microscopy (SEM) 165 After Bouin´s fixation, right holobranchs were decalcified for 6 h in a 10% 166 ethylenediaminetetraacetic acid (EDTA) solution (Osteodec, Bio‐optica®). Decalcified 167 holobranchs and remaining organs were processed for histopathology by routine methods and 168 sections (3 μm) from paraffin‐embedded tissue were stained with hematoxylin and eosin (H&E). 169 Gill tissue was additionally stained with periodic acid-Schiff (PAS) and Masson-Goldner 170 trichrome. Slides were observed and photographed using an Olympus® BX51 light microscopy 171 equipped with an DP72 or EP50 digital cameras. On the other hand, samples for SEM were 172 dehydrated in ethanol solutions and routinely processed for examination under a JEOL JSM- 173 6360LV scanning electron microscope. 174 2.6.Statistical analysis 175 The quantitative variables, body condition and opercular rate, were statistically compared 176 between groups employing the Wilcoxon-Mann-Whitney U test. The strength of the relationships 177 between body condition and opercular rate with the parasitic loads were calculated during the first 178 three samplings (day 203, 221, 225 PE) employing the Spearman´s Correlation Coefficient. In 179 addition, the daily length of the recently detached juveniles was included into the study. The 180 significance level was 95% in all cases (p-value < .05). All the numeric data were analyzed by 181 RStudio software (R Core Team, 2019). 182 3 RESULTS 183 During the late glochidiosis, no mortalities were observed and fish displayed a mean body 184 condition of 0.98 ± 0.08 and a mean opercular rate after hand-netting of 135 ± 13, with similar 185 values between the infested and the control group throughout most of the samplings (Figure S1). 186 Moreover, the juvenile detachment was successfully synchronized between day 203 and 238 PE, Page 6 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 7 187 yielding a total of 13,891 juveniles of M. margaritifera. During this period, the detachment 188 displayed a left-skewed distribution with a peak of 38 detached juveniles/fish at day 226 PE 189 (Figure 1a). Based on the curve of this detachment, three stages could be established to better 190 describe the evolution of the juvenile detachment in relation with the fish parasitosis and the 191 histopathological changes (Figure 1a and b): the rising detachment stage (day 203–226 PE), the 192 declining detachment stage (day 227–238 PE) and the post-detachment stage (day 239–246 PE). 193 3.1. The rising detachment stage (day 203–226 PE) 194 At the necropsy, 96% of fish were infested (Figure 1b) and harbored a median parasitic load 195 of 22.4 ± 47.4 larvae/fish g, in which highly infested fish in the upper quartile showed a mean of 196 102 larvae/fish g; meanwhile, the remaining fish below the upper quartile displayed a mean of 197 12.6 larvae/fish g (Figure 1c). No significant relationships were detected in the correlation 198 analysis between the parasitic load and the body condition nor the opercular rate after hand- 199 netting (Figure 1c). 200 By naked eye visualization of infested fish, abundant, well-delimited, punctiform nodules 201 were observed in the branchial tissue (Figure 2a). By stereomicroscopy, the immersed gills 202 revealed a high number of ivory bean-shaped parasitic nodules located on the four holobranchs 203 (Figure 2b), mostly at the trailing edges of the filaments (Figure 2c). Fish with less than 58 204 larvae/fish g exhibited a distinctive distribution of the larvae, clustered in bunches, which tended 205 to gather at the dorsal and ventral regions of each holobranch (Figure 2d). 206 By light microscopy at low magnifications, the parasitic clusters were composed of several 207 large and protruding nodules. Each nodule corresponded with a cyst of M. margaritifera larva, 208 surrounded by a well-localized epithelial response in which adjacent lamellae, and sometimes the 209 adjacent filaments, were fused obliterating the gill exchange surface (Figure 2e and f). Often, 210 fused lamellae became elongated up to twice their length (Figure 2f and 3c). The parasitic cysts 211 were completely covered by an intense epithelial hyperplasia and hypertrophy, the latter 212 characterized by the high number of epithelial cells with large swollen cytoplasm, ovoid nucleus 213 and small nucleoli (Figure 2g). Mitotic figures were frequently detected all over the cyst, although 214 they were more abundant basally at the interlamellar epithelium of the parasitized regions (Figure 215 2f). The epithelial cells closest to the bivalve periostracum concentrically arranged and became 216 thinner, elongated and intensely eosinophilic with H&E, being also PAS-positive (Figure 2g). 217 Associated to the parasitosis, a lymphocytic inflammatory infiltrate was interspersed between the 218 hyperplastic epithelium (Figure 2g). Moreover, a mixed inflammatory cell population, composed 219 by macrophages and polymorphonuclear cells, was identified at the deeper layers of the filaments 220 and related to the interlamellar system, particularly underneath the basal membrane of small Page 7 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 221 ladder-like vessels (Figure 2h) and marginated over the endothelium of elongated sinus (Figure 222 2i). 223 No other lesions were recorded in other sampled organs from infested fish. On the other hand, 224 control fish exhibited unfused lamellae, intraepithelial lymphocytes at the trailing edge of the 225 filaments and scarce inflammatory cells associated to the interlamellar system (Figure 2j). 226 Some parasitic nodules bulged to a high extent on the gill surface (Figure 3a), molding the 227 contiguous filaments observed as focal areas with concave shape (Figure 2e). Furthermore, fresh 228 microscopy highlighted the undisturbed arterioles underneath the parasitic cyst (Figure 3b) and 229 exceptionally devious arterioles were related to the encysted larvae at the filamental tips (Figure 230 3c). In these cases, the growth plaque of the filament was also deviated (Figure 3d) and the 231 cartilage ray was thickened due to an irregular disposition of hyperplastic chondrocytes, observed 232 by histology (Figure 3e). Under SEM, the nodular surface was covered by flattened and polygonal 233 epithelial cells, overlaid by well-defined microridges (Figure 3f). However, at the most protruding 234 regions, a localized epithelial degeneration occurred, characterized by the presence of faint 235 microridges and cell boundaries (Inset, Figure 3f). Histologically, the contact area between these 236 larger nodules and the filaments became reduced (50–100 μm width) creating a pedunculated, 237 teardrop shaped structure supported by the hyperplastic epithelium of fused lamellae (Figure 3g). 238 Occasionally, the encysted parasites were barely linked to the gill tissue by a very constricted and 239 pinched-off peduncle which showed a superficial goblet cell hyperplasia (Figure 3h). Moreover, 240 at deeper layers, the hyperplastic tissue was accompanied by moderate epithelial apoptosis, 241 observed by pyknosis, cell shrinkage and the presence of apoptotic bodies (Figure 3h). Based on 242 the overall morphopathological features described, larvae sloughed from the gill by a droplet 243 detachment mechanism, illustrated in Figure 3i and simplified in three sequential steps: 1. 244 protrusion over the surrounding limits, 2. pinch-off by reduction of the contact area between the 245 gill and 3. detachment of the larvae without tissue disruption. 246 The encysted larvae displayed a thin and refringent periostracum under fresh microscopy, 247 which allowed to discern the valves completely closed, the valve rims facing each other and the 248 discernible mantle within the internal pallial and extrapallial cavities (Figure 3j). Once detached 249 from the gills, the recently sloughed and viable juveniles exhibited mobile valves and a protractile 250 foot protruding out of the valve limits (Figure 3k). 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A., Powell, M. D. & Nowak, B. F. (2001). Ionic Effects of Infection of Ichthyophthirius 636 multifiliis in Goldfish. Journal of Aquatic Animal Health, 13(1), 20-26. doi: 10.1577/1548- 637 8667(2001)013<0020:IEOIOI>2.0.CO;2 638 Vaughn, C. C. (2017). Ecosystem services provided by freshwater mussels. Hydrobiologia, 810(1), 639 15-27. doi: 10.1007/s10750-017-3139-x 640 Wächtler, K., Dreher-Mansur, M. & Richter, T. (2001). Larval Types and Early Postlarval Biology 641 in Naiads (Unionoida). In G. Bauer & K. Wächtler (Eds.), Ecology and Evolution of the 642 Freshwater Mussels Unionoida (Vol. 145, pp. 93-125): Springer Berlin Heidelberg. 643 Wacker, S., Larsen, B. M., Karlsson, S. & Hindar, K. (2019). Host specificity drives genetic 644 structure in a freshwater mussel. Scientific Reports, 9(1), 10409. doi: 10.1038/s41598- 645 019-46802-8 646 Waller, D. L. & Mitchell, L. G. (1989). Gill tissue reactions in walleye Stizostedion vitreum vitreum 647 and common carp Cyprinus carpio to glochidia of the freshwater mussel Lampsilis 648 radiata siliquoidea. Diseases of Aquatic Organisms, 6(2), 81-87. doi: 10.3354/dao006081 649 Wootten, R. (1974). The spatial distribution of Dactylogyrus amphibothrium on the gills of ruffe 650 Gymnocephalus cernua and its relation to the relative amounts of water passing over 651 the parts of the gills. Journal of Helminthology, 48(03), 167-174. doi: 652 10.1017/S0022149X00022793 653 Page 18 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 19 654 Figures 655 Figure 1. (a and b) Evolution of the mussel juvenile detachment (a) in relation with the fish 656 parasitosis (b) during the three stages of late glochidiosis of M. margaritifera delimited by dashed 657 red lines. (c) No correlation between the parasitic loads and the fish body condition nor the 658 opercular rate were detected in the correlation analysis. Individuals with parasitic loads in the 659 upper quartile were represented in red. Confidence interval was 95%. 660 Figure 2. Main morphopathological findings during the rising detachment stage of late M. 661 margaritifera glochidiosis. (a) Gill macrophotography of an infested fish with punctiform lesions 662 which corresponded with the encysted larvae (arrows). (b and c) Stereomicrophotographs 663 showing the bean-shaped larvae located between each pair of hemibranchs indicated with 664 arrowheads. (d) Distribution of parasites clustered in bunches at the dorsal and ventral regions of 665 each holobranch (asterisks). (e) The localized epithelial response enclosing the parasitic cysts was 666 accompanied by severe lamellar and filamental fusions (asterisks). Note the concave depressions 667 on the filament surface contiguous to an adjacent larva (arrows). H&E stain. (f) Surround the 668 outer surface of the parasitic cyst (arrowheads), the fused and elongated lamellae (dashed lines) 669 were related to abundant mitotic figures (circles) and interlamellar cysts (arrow). Note the 670 pleomorphic inflammatory infiltrate located at the interlamellar system (asterisks). H&E stain. 671 (g) A high number of lymphocytes interspersed between epithelial cells, the latter became 672 flattened and eosinophilic towards the larvae (arrowheads). H&E stain. Inset: The eosinophilic 673 epithelial cells (arrows) beside the parasitic cysts (arrowheads) became thinner and more intensely 674 stained under the PAS stain. (h) Interlamellar system with a pleomorphic inflammatory (asterisks) 675 underneath the intricate basement membrane highlighted with the Masson-Goldner trichrome 676 stain. (i and j) Comparison between infested fish with marginated macrophages and 677 polymorphonuclear cells over the interlamellar system endothelia (arrowheads, i) and control fish 678 showing a normal lamellar morphology without leukocytes over the endothelial cells lining the 679 interlamellar system (arrowheads, j). H&E stains. 680 Figure 3. Gill morphopathology of the most superficial parasitic cysts during the rising 681 detachment stage of late M. margaritifera glochidiosis (a-h) and the droplet detachment (i) from 682 encysted larvae into free-living juveniles (j and k). (a) Scanning electron microscopy (SEM) of a 683 cluster of buoyant encysted larvae (asterisks). (b and c) Fresh microphotographs comparing the 684 unaltered arterioles (b, arrowheads) with exceptionally devious arterioles (c, arrowheads). (d and 685 e) Deviation of the growth plaque (arrowheads, d) and a well-localized cartilage hyperplasia 686 (arrowheads, e) overgrowing over the cartilage ray (asterisk, e). H&E and Masson-Goldner 687 trichrome stain, respectively. (f) SEM of a parasitic cyst completely covered by epithelial cells 688 with the dashed area highlighting the most protruding area. Inset: Detail of the dashed area with Page 19 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 689 poorly discernible microridges over the epithelial surface (arrowheads). (g) Histological 690 microphotograph of the hyperplastic peduncles (asterisk) anchoring the larvae to the gill 691 filaments. Note the presence of an elongate lamella (dashed line). H&E stain. (h) Constricted 692 peduncle with a high number of goblet cells lined up the epithelial surface (asterisks) and high 693 number of apoptotic (arrowheads) and phagocytic bodies (arrows) at deeper regions. PAS stain. 694 (i) Schematic illustration of the three-steps droplet detachment of the mussel juveniles. (j) Fresh 695 light microscopy of an encysted larvae with the valve rims facing each other (arrowheads) and 696 their internal pallial cavity (asterisk). (k) Stereomicrophotograph of recently detached and viable 697 juveniles with their protractile foot (arrowheads). 698 Figure 4. Fusions and interlamellar cavities adjacent to the encysted larvae during the rising 699 detachment stage of late M. margaritifera glochidiosis. (a) The limits of the interlamellar cavities 700 were barely visible by fresh microscopy (arrowheads) close to the parasitic cysts (asterisks). Inset. 701 Detail of an interlamellar cyst associated with two fused lamellae (arrowheads). (b) Solid lamellar 702 fusion (dashed lines) associated with the presence of abundant interlamellar mitotic figures 703 (encircled) and goblet cells at the surface (asterisks). PAS stain. (c and d). Partially fused lamellae 704 characterized by the presence of interlamellar cysts (asterisks) and clefts (arrowheads) surrounded 705 by mucous cells. PAS stains. (e) SEM of two fused filaments (dashed line) with small openings 706 located between partially fused lamellae (arrowheads). (f) Detail of the dashed area highlighting 707 two interlamellar openings (asterisks) lined up by epithelial cells. 708 Figure 5. Gill morphopathology during the declining detachment stage (a–h) and the post- 709 detachment stage of late M. margaritifera glochidiosis (i–k). (a) Stereomicroscopical image of 710 disordered lamellae observed by tousled secondary filaments (arrowheads). (b) By histology, 711 altered lamellae ranged from hyperplastic and fused lamellae (asterisks) to elongated and bent 712 lamellae (arrowheads). H&E stain. (c) Detail of elongated lamellae with their tips bent inwards 713 and slightly hyperplastic (arrowheads). Note the presence of a moderate number of pleomorphic 714 inflammatory cells at the interlamellar system (asterisks). H&E stain. (d) Hyperplastic and fused 715 lamellar tips (arrowheads) giving rise to an elongated interlamellar cavity (asterisk). H&E stain. 716 (e) Detail of hyperplastic lamellae with most of the goblet cells arranged towards the interlamellar 717 clefts (arrowheads) with openings towards the surface (arrowheads). PAS stain. (f) Partially fused 718 lamellae showed areas of moderate lymphocytic (asterisks) and localized areas of epithelial 719 degeneration (arrows). H&E stain. (g) Detailed microphotograph of abundant apoptotic bodies 720 (asterisks) and vacuolized, swollen and sloughing epithelial cells at the outer surface 721 (arrowheads). H&E stain. (h) Phagocytosis of apoptotic bodies (arrowheads) and small PAS- 722 positive vacuoles (arrows) was associated with an area of lamellar fusion. PAS stain. (i and j) 723 Normal structure of outer surface of the gill by stereomicroscopy (i) and scanning electron Page 20 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy 21 724 microscopy (j). (k) Histological photograph showing mild epithelial hyperplasia, elongations and 725 lamellar deviation (arrowheads) together with a moderate number of mononuclear cells at the 726 interlamellar system (asterisks). H&E stain. 727 Supplementary table and figure are included in Supporting Information. 728 Page 21 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy Page 22 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy Page 23 of 27 Journal of Fish Diseases Journal of Fish Diseases Review Copy Page 24 of 27 Journal of Fish Diseases Journal of Fish Diseases