1 Microscopic and submicroscopic observations of the mantle and gills of oysters

2 (Crassostrea hongkongensis) infected with Polydora

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4 Chang-Yu Guo a,b ,Jiang-Yong Wang a,* ,JiaHao-Zhaoa,b, Kai-Yuana ,
5 JingFeng-Sunb* , ZhaoRui-Wanga,*
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7 aCollege of Life Sciences, Huizhou University, Huizhou 516007, China
bCollege of fisheries, Tianjin Agricultural University, Tianjin

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9 Xiqing District 300384, China)
10 *Corresponding author: wjy104@163.com
11

12 ABSTRACT

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13 The abnormal symptoms and the pathogenic mechanism of the Hong Kong oyster
14 (Crassostrea hongkongensis) infected with the shell-boring polychaete Polydora
15 ciliata. Mantle and gill tissues were sampled from both infected and healthy oysters
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16 and analyzed using transmission electron microscopy. Infected oysters displayed
17 symptoms such as black mud tubes filling the inner and outer surfaces of the shell,
18 ulceration of the soft body, darkening of the color, atrophy and erosion of the mantle
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19 and other tissues, and an increase in mucus. Micropathological changes included
20 tearing of connective tissue, dissolution, disappearing crest protrusions, an increase in
21 secretory cells, vacuolizationvacuolization of cells, nuclear membrane rupture,
22 contraction and dissolution of nuclei, and partial or complete disappearance of
23 organelles. Additionally, the secretory cells of infected individuals displayed
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24 disorganized distribution and nuclear aberrations, with some secretory particles

25 overflowing.
26
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27 Key Words: Oysters; Crassostrea hongkongensis; Polydora, mantle; gill; adductor

28 muscle; transmission electron microscope

29
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30 1. Introduction

31 In 2021, the annual output of oysters will exceed 5 million tons, accounting for
32 40% of the output of Mollusca and 70% of the world's total output. The Hong Kong
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33 oyster Crassostrea hongkongensis, also known as "white oyster," is commonly

34 cultured in South China, primarily in the coastal areas of Guangxi, Guangdong, and
35 Fujian (Wang et al., 2007). Methods includeintertidal planting , hanging raft , trellis
36 pond, and other methods. The oyster is one of the main molluscs mollusccultured on
37 south China coast with large scale and high economic value (Zhang et al., 2012).
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38 The oyster industry is, however, often impacted by diseases, one of which is caused
39 by Polydora spp., a parasitic worm that burrows into the oyster shell and causes
40 significant damage to the shell structure and soft tissue. Due to the limitations

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 41 oftechnology, early research on diseases was limited to parasites. After the 1970s,
42 mollusccan diseases caused by bacteria and viruses were gradually recognized and

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43 valued (Wang, 2018). The Polydora complex, which is affiliated with the Annelidae,
44 Polychaete, Cryptozoospermia, Spionidae, is a collective name for all species of

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45 Spionidae that have metamorphosed in the fifth ganglia (Lauckner, 1983), with a
46 total of more than 140 species. They can spread globally with ocean currents in the
47 larval stage, and the feeding mechanism of polydorais flexible and adaptable to
48 complex and changeable environments, not only in soft sediment or silt layers but
49 also in hard limestone materials, such as marine corals, coral algae, and molluscan
50 shells, such as oysters, abalone, mussels, and scallops (Gao, 2011). Most Polydora

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51 inhabit near-shore waters, such as intertidal zones, bays, and estuaries., and some of
52 them are common polychaete parasites on mollusccs wherethey burrow into the
53 shells and produce dark mud bubbles or argillaceous tube holes inside their host
54 shells (BLAKE , 1971; BLAKE , 1983; MACIOLEK , 1984), commonly known as
55 "mud worms" or "mud bubble worms" (Blake, 1969). Thea planktonic larval stage

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56 begins to sink when it reaches the sixteenth ganglion planktonic larvae and then
57 parasitizes on the outside of the calcareous shell (Loosanoff and Engle, 1943;
58 Clements et al., 2018), peristalsing to form a U-shaped pipe with two external
59 openings (Korringa, 1954). As the larvae of Polydora continue to creep, the resulting
60 pipe enters the inner surface of the shell, causing the host to produce one or more
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layers of thin nacre to isolate the pipe on the outside (Haigler, 1969; Zottoli, 1974;
Xie, 1995; Handley, 1997). The larvae continue to expand the pipe burrows under the
63 thin calcareous layer produced by the host, forming "mud bubbles" as the space is
filled with debris, dirt, and larval droppings (Zottoli, 1974; Morse et al., 2015). Its
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65 irregular shape and darker color affect the appearance of oysters. The Polydora
66 parasitizes in the shell of molluscs, leading to damage and deformation of the host
67 shell, seriously damaging shell components, and even damaging the soft tissue part
68 of molluscs, causing local inflammation, ulceration and atrophy (Gao, 2011).
69 Thishindersgrowth and development of the host molluscs, leading to a significant
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70 decline in quality, and often to high mortality in severe cases (Waka et al., 2016),
71 making this parasite a serious impediment to the development of molluscan
72 aquaculture.
73 The Polydora complex has spread widely and has seriously damaged the oyster
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74 culture industry worldwide. Studies on the impacats of Polydora ciliata on scallops,

75 pearls, variegated baumannia, and oysters have been reported (Sun et al., 2002; Ren
76 et al., 2003; Chen et al., 2004), previous studies (Liu et al., 2020; Shi et al., 2012; Du,
77 1999) have shown that different regions of the mantle may correspond to different
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78 biological functions during shell formation. The infection rates of polychaete

79 parasitic diseases in Fangchenggang City, Beihai City, Shenzhen City, Guangdong
80 Province and Lingshui County, Hainan Province, were all over 76% (Shi et al., 2004).
81 The high mortality rate of the Japanese scallop (Mizuhopecten yessoensis) (Jay 1857)
82 in British Columbia, Canada, is attributed to Polydora websteri. For instance, in
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83 British Columbia, P. websteri caused up to 84% mortality in scallop grow-out sites

84 from 1989 to 1990, resulting in up to US $449,660 in lost revenue that year (Bower
85 et al., 1992; Shinn et al., 2015).This introduction caused significant damage to
86 mollusc production (Lunz, 1940). Since the 1940s, oyster farms on the east coast of
the United States have been plagued by Polydora websteri, resulting in significant
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88 losses of oyster farms (Bower et al., 1992). These examples illustrate the significant
89 impact of the invasion of Polydora on the local aquaculture industry. Studies have
90 shown that the mechanism of the occurrence of parasitic diseases of marine molluscs

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 91 is complex, and it is impossible to control the occurrence and epidemic of diseases
92 completely at present .

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93 The entire soft part of the oyster is included in the mantle, surrounded by two
94 pieces of mantle on the left and right to form a mantle cavity, which can form plain

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95 colors, secrete immune components, and can also carry out gas exchange, which is an
96 important organ of oysters. The gills of bivalves are respiratory organs as well as
97 filter-feeding organs. The gills of oysters are located above the visceral mass, and on
98 both sides, there is a pair of gill flaps, which are composed of numerous gill filaments
99 whose epithelium can be divided into four different regions: the columnar epithelial
100 area of the preciliate, the flat or cubic epithelial area, the lateral ciliated columnar

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101 epithelial area, and the posterior cilia columnar epithelial area. The anterior cilia on
102 the gills are filled with mitochondria, which can provide a constant source of energy
103 for gas exchange and water flow in the gills., Chlorine cells, as an important cell on
104 the gills of oysters, play an important role in the osmoregulation of bivalves in the
105 aquatic environment. Jiang Ming et al. (2001) identified chlorine cell types in Pacific

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106 oyster gills (Jiang et al., 2001) and described their ultrastructural characteristics,
107 suggesting that chlorine cells are an important cellular system for osmotic pressure
108 regulation in Pacific oysters. Thus, the lesions produced by oyster gill cells, including
109 chlorine cells, can also be used as research objects for the important impact of
110 Polydora infection on oysters.
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In this study, the microstructure and ultrastructure of the mantle and gills were
observed using optical and transmission electron microscopyto elucidate the
113 physiological basis of the influence of Polydora infection on oysters, which can
provide a scientific basis for an in-depth understanding of the effects of Polydora
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115 infection on the mantle and gills of oysters.
116 2. Materials and Methods
117 2.1. Collection and Temporary Cultivation of Experimental Oysters.
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118 Hong Kong oysters Crassostrea honkongensis were collected from Oyster Bay,
119 Tiechong Town, Huidong County, Guangdong Province, China with an average shell
120 length and width of 100 ± 10mm and 50 ± 10mm, respectively. The oysters were
121 temporarily held in the laboratory for seven days, in seawater with a salinity of 18±3.,
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122 and fed receiving twice daily. After seven days, two groups of oysters were
123 randomly divided based on their health conditions: the first group consisted of
124 healthy oysters of similar size and high viability, and the second group consisted of
125 oysters of similar size infected with Polydora, displaying more mud pipes and holes
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126 on their shells. Ten oysters were sampled every day, and the weight of healthy and
127 infected oyster soft parts and the infection intensity of infected oyster soft parts and
128 shells were recorded to observe the intuitive impact of infection intensity on oysters.
129 The weight of the software part was measured using an electronic balance with an
130 accuracy of 0.1g. The intensity of infection was determined by observing the shell
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131 and soft parts. A minor infection was characterized by a few smaller mud tubes or re-
132 covered mud tubes, with no obvious symptoms in the soft part. A moderate infection
133 was characterized by larger wormholes appearing under the fragile nacre and more
134 mud pipes, with slight atrophying and small lesions in the soft body part. A severe
135 infection was characterized by the shell being almost completely occupied by mud
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136 pipes or wormholes, with discoloration, atrophy, and ulceration of the soft body part.
137 The data are summarized in Table 1.

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 138

Table 1.

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140 Oysters Infected with Polydora in Tiechong Town
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142 Group Soft weight (g) Number of infections Infection rate (%)
143 Mild Moderate Severity

144 A 23.1±2.6 6 16.7 33.3 50.0

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145 B 26.3±2.9 5 80.0 20.0 0
146 C 25.4±1.2 6 50.0 16.7 33.3
147 D 25.8±4.7 5 60.0 40.0 0

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148 E 27.1±1.9 8 62.5 25.0 12.5
149 F 25.6±1.6 8 50.0 25.0 25.0
150 G 22.1±2.4 9 22.2 22.2 55.6
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152 As shown in Table 1, most of the Hong Kong oysters collected from tiechong
153 town were infected with Polydora. The infection rate of group B and group D was
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154 the lowest. The average body weight of these groups was higher than that of other
155 groups. The infection rate of group G was the highest, and the average body weight
156 was the lowest. The weight of the rest healthy oysters was 30.6 ± 1.2, which was
157 generally higher than that of the infected group. These results indicate that Polydora
158 infection can lead to the growth stagnation or even atrophy and dissolution of the soft
159 part of oysters, resulting in weight loss.
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160 2.2. Light microscope observation.

161 Three healthy oysters and three severely infected oysters of similar size were
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162 taken about near the edge of the mantle 10mm × 10mm tissue. They were fixed with
163 Davidson's fixative, embedded in paraffin, and stained with H.E. Finally, the samples
164 were embedded in neutral glue and observed under a light microscope.
165 2.3. Transmission Electron Microscopy.
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166 Three healthy oysters and three severely infected oysters were thinly cut, and the
167 mantle and gill margins were about 6mm ³ Of tissue blocks. Pre fixed with glass
168 dishes containing 2.5% glutaraldehyde fixative solution, washed with PBS solution,
169 stored in the dark for 2 hours at room temperature, and overnight at 4 ℃. The fixed
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170 samples were transported to Guangzhou Saville Biotechnology Co., Ltd. for routine
171 transmission electron microscope preparation, ultrathin sections (70-90 nm) were
172 stained, and Hitachi ht7700 transmission electron microscope was used for
173 observation.
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174 3. Results
175 3.1. Clinical Symptoms
176 Oysters infected with Polydora exhibit symptoms such as slow response to
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 177 stimuli, holes and mud pipes on the shell surface, blackening of the inner and outer
178 shell surfaces, mud bubbles filled with Polydora debris and feces, increased secretion

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179 of surface mucus, and dirt buildup in organs such as the mantle, gonads, and visceral
180 masses. The soft body of infected oysters appears yellowish brown or light brown

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181 (Fig. 1B), the mantle becomes thin and transparent, and the marginal membrane is
182 brownish brown. Visceral masses and gills were grayish green or grayish brown,
183 eroding some gill filaments.
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189

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191 B C
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193 Fig. 1. A. A healthy oyster shell and soft tissue; B. An oyster infected with Polydora,
194 showing changes to the shell and soft tissue; C. An oyster infected with Polydora ( →
195 Indicated polydora)
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197 3.2. Histology, Ultrastructure, and Pathological Analysis of Healthy and Infected
198 Oyster Mantle tissue
199 Under light microscopy, the marginal membrane of the mantle consists of three
200 parts: shell protrusion (Fig. 3-P1), sensory protrusion(Fig. 3-P2), and marginal
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201 membrane protrusion (Fig. 3-P3). The tissue structure includes an outer epithelial
202 layer, inner epithelial layer, muscle fibers, connective tissue, and mucus-secreting
203 cells. Healthy oysters have three main types of cells in the mantle: columnar
204 epithelial cells, electron-hyaline granulocytes, and electron-dense granulocytes, with
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205 columnar epithelial cells further divided into A, B, and C types, with cell sizes
206 between 10 and 15 μm. In healthy individuals, type A cells dominate the marginal
207 membrane, type B cells dominate the central membrane, and columnar epithelial cells
208 are evenly arranged in the marginal membrane. The basement membrane serves as a
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209 base for type A cells, with type B cells primarily located underneath. Type A and B
210 basement membranes interpenetrate, forming an irregular and linear “membrane
211 labyrinth” structure.
212 3.3. Histology
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213 Tissue section staining of infected oyster mantles shows a reduction in crest-like
214 protrusions on the marginal membrane and decreased epithelial cell surface area, with
215 an increase in secretory cells, while there were a large number of basophilic secretory
216 granules in the marginal membrane epithelial layer of the mantle of healthy
individuals, dark blue; a few weak basophilic secretory granules were light blue or
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218 gray, and in the connective tissue layer adjacent to the epithelial layer, the granules
219 secreted by eosinophils were red or bright red (Ren et al., 2003). In healthy
220 individuals, the distribution of secretory cells and granules is orderly, with a layer of

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 221 secreted mucus evenly distributed over the epithelial layer (Fig. 2A). In infected
222 individuals, the distribution of secretory cells is disordered, with severe intracellular

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223 vacuolization, nuclear aberrations, partial overflow of secretory granules, mucus
224 masses attached to the outer epithelial surface, and an overall increase in secretory

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225 cells, including eosinophils and basophils (Fig. 2B).
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SC1
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234 SC2
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236 SC2
237 SC1
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50μm 50μm
239
A B
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242
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Fig. 2. A. Partial structure of the epithelial layer of the shell protuberance of the mantle in
243 healthy oysters; B. Structure of the epithelial layer of the shell protuberance of the mantle in
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244 an oyster infected by the Polydora. Key: SC1: Eosinophilic secretory cells, SC2: Basophilic
245 secretory cells. Bar = 50 μm.
246
247 In healthy individuals, the connective tissue and muscle fibers of the marginal
248 membrane have a certain regularity, with muscle fibers gradually increasing and
connective tissue gradually decreasing towards the tip of the protrusion (Fig. 3B). In
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250 infected individuals, connective tissue is denser and similar to that at the base, with
251 band-like vacuoles in the shell protrusion and secretory cells close to the connective
252 tissue layer that have shed into the connective tissue. The outer epithelium of healthy
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253 individuals' marginal membrane protrusions has crest-like protrusions with evenly
254 distributed internal structure, while in infected individuals, the outer epithelium of
255 marginal membrane protrusions has no obvious crest-like protrusions, with only
256 crest-like protrusions below the bottom of the protrusions and torn and dissolved
257 muscle fibers and connective tissue (Fig. 3A).
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258
IEP
259 P3
CT
CT
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260 MF
MF P3
261 MF MF
P2
262 CT
P2 P1
OEP
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263 CT
264 OEP
A 200μm P1 400μm
B
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 266 Fig. 3. A. Rimmed membrane protrusions of the mantle in an oyster infected by the Polydora;B
267 . Rimmed membrane protrusions of the mantle in healthy oysters. Key: P1: Shell protrusion,

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268 P2: Sensory protrusion, P3: Marginal membrane protrusions, IEP: Inner epidermis, OEP:
269 Outer epidermis, CT: Connective tissue, MF: Muscle fibers. Bar = 200 μm.
270

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271 3.4. Ultrastructural observations
272 Electron microscopy images of healthy oysters showed that the epithelial layer
273 of the mantle contained a large number of A-type columnar epidermal cells (Fig. 4A),
274 some arranged in a monolayer and some scattered throughout the layer. The cells

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275 were about 7 to 14 μm in length, with a nucleus that occupied most of the cell. The
276 nuclei had distinct autochromatin and heterochromatin, and were mostly located in
277 the middle and lower parts of the cells. The organelles were relatively intact. After
278 infection by Polydora (Fig. 4B), the overall image of the oyster mantle appeared
279 darker than that of healthy individuals, with a particularly prominent staining of the
280 epithelial layer area. This was due to the abnormal increase of secretion granules and

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281 pigment particles in the epithelial layer caused by the Polydora infection. The HE
282 staining also confirmed this, as the pigment particles and secretion granules in the
283 epithelial layer area of the marginal membrane protrusion of the infected individual
284 were significantly increased, and the internal connective tissue area was significantly
darker than that of healthy individuals.
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287
CE-A
288
Mv
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BM
293 CE-A
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CE-A
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295 lkk
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297 20μm 20μm
A B
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300 Fig. 4. A. Ultrastructure of marginal zones of healthy oyster mantle; B. Ultrastructure of
301 marginal zones of diseased oyster mantle; BM: basement membrane, CE-A: columnar
302 epidermal cell type A, Mv: microvilli, bar=20 μm.
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304 Below the basement membrane, there was some disordered white zona pellucida
305 (Fig. 5),
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20μm
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 316
317

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318 Fig. 5. Ultrastructure of the marginal zones of the mantle in oysters showing lytic-like
319 lesions. Bar=10 μm.

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321 consisting of electron-transparent granulocytes and secretory cells, that flooded
322 between the epithelium and connective tissue layers. The overall structure inside the
323 marginal membrane of healthy oysters was clear, with a normal nucleus morphology,
324 complete nuclear membrane, and no chromatin shrinkage or vacuolization inside the
325 nucleus. The outer mitochondrial membrane was clear and the inner crest was

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326 relatively clear. Lysosomes were small, with a clear outer membrane and
327 homogeneous internal substance, and the endoplasmic reticulum was laminated and
328 relatively normal (Fig. 6A).
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330

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332 G

333 N
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M N
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336 M
337 2μm
338 A 2μm B
339
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340 Fig. 6. A. Ultrastructure of healthy oyster mantle cells; B. Ultrastructure of diseased oyster
341 mantle cells; N: nucleus, M: mitochondrion, G: Golgi apparatus, bar = 2 μm.
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343 In infected individuals, cells showed obvious pathological changes, with small
344 fractures in the nuclear membrane, chromatin shrinkage, and vacuolization in the
nucleus. A large part of lysosomes, the Golgi apparatus, endoplasmic reticulum, and
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346 mitochondria had dissolved and disappeared, and the only remaining morphological
347 structure of the endoplasmic reticulum and cellular mitochondria was not obvious,
348 showing semi-degenerate and dissolved forms. The cytoplasm also appeared lysed
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349 and there was severe vacuolization (Fig. 6B).

350
351 3.5. Ultrastructural and Pathological Analysis of Healthy and Infected Oyster Gills
352 The gills of healthy oysters have densely packed cells and contain normal levels
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353 of secretions without any other contaminants (Fig. 7A). Electron microscopy showed
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356
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357 M
358 N
359
Mv Mv
360
361 RER
G
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362 M N
363
5μm 5μm
364 A B
365

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 366 Fig. 7. A. Ultrastructure of healthy oyster gill cells ( → Indicated lesions location); B.
367 Ultrastructure of diseased oyster gill cells; N: nucleus, M: mitochondrion, G: Golgi apparatus,

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368 Mv: microvilli, RER: rough endoplasmic reticulum, bar = 5 μm.
369

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370 the three main cell types in oyster gills: cubic cells, ciliated cells, and glandular cells.
371 Ciliated cells have abundant mitochondria, rough endoplasmic reticulum, and
372 developed Golgi apparatus. The mitochondria are round or oval and mainly located
373 near the nucleus and tip of the cytoplasm, and have a high number of microvilli and
374 some visible microtubule structures. The surface of cubic cells is covered with dense
375 microvilli and the nucleus takes up most of the cell. Glandular cells have several cell

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376 types and their nucleus is at the base of the cell, with a large number of transparent
377 dense particles and vacuole-like substances in the cytoplasm, Meanwhile an
378 aggregated "bacterial mass" was found in the glandular cells of infected oysters, and
379 the outer layer was surrounded (Fig. 8).
380

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389 2μm
A 2μm
390 B
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Lf
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Cc
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5μm 5μm
394
C D
395
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396 Fig. 8. A. Ultrastructure of healthy oyster glandular cells; B. Ultrastructure of diseased oyster
397 glandular cells (→Indicated bacterial colony); C. Cuboidal cell zone; D. Ciliated cell zone; N:
398 nucleus, Cc: Cuboidal cell , Lf：ciliated cell, bar = 5 μm.
399
400 The gill tissue of infected oysters is close to dissolving and collapsing, and the
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401 damaged tissue is surrounded by interstitial fluid containing various secretory cells,
402 organelles, and microvilli. The nucleus of pregill leaf cilia cells, gill leaf cubic cells,
403 and gill epidermal cells have abnormal nuclear karyotype, multiple invaginations of

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 404 nuclear membranes, partial nuclear solidification, and ruptures of nuclear membranes,
405 and some organelles such as the Golgi apparatus are missing. Some cells also have

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406 dense microvilli that have collapsed (Fig. 7B).
407 3.6. Possible pathogenic and lethal factors for infected oysters

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408 Most organelles in all mantle and gill tissues of diseased oysters, showed
409 irreversible damage to various degrees, in mitochondria, lysosomes, and endoplasmic
410 reticulum.The damage of organelles must lead to abnormal cell function, as
411 mitochondrial damage will affect energy supply, intracellular ion balance is disrupted,
412 causing edema of cells and intracellular organelles, mitochondrial membrane rupture,

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413 degeneration; Lysosomes lyso - somes secrete decreased function, unable to timely
414 clear harmful substances from the pericapsule, causing accumulation of intracellular
415 inflammatory substances, which leads to the moribund collapse of cells, further
416 resulting in whole tissue lesions, ulcerated atrophy of the diseased oyster muscle, and
417 loss of ability to eat and act until death from systemic failure.

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418
419 4. Discussion
420 Parasitism by Polydora in molluscs and the resulting decline or death is linked to
421 specific cell types within the cells. For instance, the damaged organelles and cell types,
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422 such as the mitochondria, chlorine cells, and glandular cells, in diseased Haliotis
423 diversicolor individuals result in reduced energy provision, the Na + - K + balance in
424 the cell is disrupted, causing intracellular Na + stores, which allow excess extracellular
425 water to enter the cell,causing cell swelling, organelle structure abnormalities and even
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426 death (Chen et al., 2004). The present experiment showed that the glandular cells,
427 chlorine cells, and various organelles in oyster tissue infected by the Polydora were
428 abnormal, hindering normal physiological functions.
429 There are conflicting opinions on the pathogenicity and lethality of
430 microorganisms that parasitize marine molluscs (Ren et al., 2002). While some reports
suggest that the death of pathogenic molluscs is linked to infection by pathogenic
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432 organisms (Wu et al., 1995; Gulka et al., 1983; Norton et al., 1993; Wen et al., 1994),
433 others argue that pathogenic organisms cause minor or no damage to host tissue cells
434 (Elston, 1986; CAROL and G, 1983; Renault, 1994). The data prsented here showed
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435 minor pathological changes in oysters with mild infections and no obvious changes in
436 the soft body part, while severely infected individuals had obvious morbidity, serious
437 tissue cell destruction, and lost some physiological functions, resulting in death. Hence,
438 the harm caused by the Polydora infection to oysters depends on the degree of
439 infection, and severe infection can cause serious pathological damage or death to the
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440 host.
441 Environmental conditions (mainly temperature and salinity) play an important
442 role in regulating parasite pathogenicity and host disease resistance (Liang et al.,
443 2003). Liu et al. (2021) identifieda new Hong Kong oyster parasite, Thraustochytrium
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444 pachydermum, at salinitiesbetween 15 and~25. The salinity of the environment affects

445 the proliferation and size of T. caudivorum in Hong Kong oysters, with the highest
446 number of zoospores produced at 25 salinity (Liu et al., 2021). Similarly, Tsui et al.
447 (2012) found that salinity affects the yield and motility of zoospores in four Mangrove
448 Thraustochytrid isolates, each of which had its optimal range of proliferative salinity
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449 (Clement et al., 2012), indicating that salinity is an important factor affecting parasite
450 proliferation and infection pathogenicity. The increase of silt in the environment can
451 also increase the susceptibility of oysters to Polydora (Clements et al., 2018), while a

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 452 decrease in pH value can weaken it (Clements et al., 2017). The number of oysters
453 infected with Polydora also shows a cyclical periodicity. Recently, through collection

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454 and dissection experiments of Crassostrea hongkongensis, it was found that the
455 number of oysters infected with Polydora showed cyclical periodicity, and the number

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456 of infected Polydora in each batch of collected oysters fluctuated. The less infected
457 individuals did not have obvious abnormalities. In order to ensure the accuracy of the
458 experiment, this experiment synthesized the results of several sampling, and selected
459 the oyster with the highest degree of infection for experimental analysis.
460 Through the sectional observation of oyster mantle, gills and other tissues, it was
461 found that the pathological symptoms of oysters infected with Polydora are similar to

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462 those of other parasites such as Perkinsus, including disordered and dissolved
463 connective tissue and muscle fibers in the mantle, increased dirt and secretion in the
464 gills, dull soft body parts, emaciation and ulceration, growth retardation, and reduced
465 reproductive capacity. In the early stage of parasite infection, inflammation occurs
466 only in the tissues around the pathogen, and the secretion increases. In the later stage,

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467 most tissues will be infected to varying degrees. After oysters are infected with
468 Perkinsus marinus, it is extremely emaciated, meat is loose, digestive glands are dark
469 gray, mantle shrinks, gonad development is blocked, growth is slow, and sometimes
470 abscesses appear. (Liang et al., 2003). After P. marinus invades the oyster shell
471 through the digestive tract epithelium, the primary lesion is established in the shell,
472
473
er
causing extensive abscess in the epithelial layer, epithelial cells and infiltrated blood
cells are destroyed, and the worm body reaching the connective tissue reaches the
474 whole body tissues and organs through the blood vessels and causes multiple
abscesses in the connective tissue of various organs, especially the mantle. Gao Yan
pe
475
476 found that Polydora ciliata, caused oyster to become brittle, forcing shellfish to
477 consume more energy to form shellfish to resist the invasion of polydora, rather than
478 for their own growth and development，and at the same time the shellfish are more
479 susceptible and have increased mortality due to the large amount of microorganisms
480 contained in flocculent debris in the duct of the chilblains. Diseased oysters manifest
ot

481 with weight loss, growth retardation or cessation, and reduced reproductive capacity
482 (Ray and Chandler, 1955). In 2021, the oyster parasite Thraustochytriu. caudivorum
483 was first isolated from the gill tissue of crassostrea hongkongensis, and the flesh of
infected oysters turned black, dissolved and even lead to death (Liu, 2021).
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484
485 During the microscopic examination of oyster tissues in this study, numerous
486 mucus cells were found. Although commonly found in molluscs, mucus cells are not
487 well-studied and are still being explored. Mucus cells play a crucial role in fish
488 immunity (Ren et al., 2003). AB-PAS staining (Sun, 2002) showed the presence of
rin

489 various mucus cells on the marginal membrane cells of molluscs, and many RNA, acid
490 polysaccharides, neutral polysaccharides, and high ACPase activity were found on
491 mucus cells of molluscs such as Chlamys farreri, Nuttallia olivacea, and Meretrix
492 meretrix (lv, 2004). ACPase, an important hydrolase in animals and a marker enzyme
493 for lysosomes, suggests that the mantle mucus cells in molluscs, including oysters,
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494 have some immunoprotective functions. Abnormal enzyme activity is one of the
495 features of histopathological changes (Shao, 1995). Diseased molluscs may experience
496 decreased digestive function and metabolic disorders, leading to extensive damage to
497 the tissues and organs. Zhou et al. proposed that marine molluscs resist foreign
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498 pathogens through the combined action of cytokines (such as blood cells) and various
499 humoral immune factors (such as lysosomal enzymes) (Nell and Nith, 2000). This is
500 supported by the presence of eosinophilia at the end of the mantle protrusion after
501 oyster infection, as eosinophils are blood cells that mediate cellular immune responses

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 502 in parasitic infections, indicating that infected oysters activate immune function by
503 increasing cytokines such as blood cells.

d
504
505 5. Conclusions

we
506 In this study, the pathological changes produced in the mantle and gills on
507 microscopic as well as submicroscopic structures of oysters infected with Polydora
508 wereinvestigated and the pathogenic mechanism of Polydora infection elucidated.It is
509 clear that Polydora causes damage to the tissue cells by infestation of its crust, the
510 mollusc, and then by local extension to the tissue cells of the entire oyster, causing the

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511 shell of the oyster to become full of cavities, leading to shrinkage and ulceration of the
512 mollusc, and even death. The microscopic structure mainly show disordered secretion
513 and abnormal rupture of the muscle connective tissue by the appearance of mantle
514 protrusion structures, and submicrostructurally, occur with a high frequency as
515 organelle abnormalities or disappearance. Taken together, the above experimental

re
516 studies proved that after oysters were infected with Polydora, the parasitic behavior of
517 Polydora caused shell damage and accumulated large amounts of pathogenic
518 microorganisms, resulting in irritant- producing inflammatory lesions on individual
519 oyster tissues. When stimulation exceeds the level of self tolerance in oysters, it can
520 cause host death. Based on the microstructural images, as well as ultrastructural
er
521 images, it can be inferred that Polydora infection can lead to cross infection with
522 various pathogenicmicroorganisms, loss of host immunity, abnormal phagocytosis of
523 self cells such as lysosomes and phagophores, and other organelles, leading to
524 incomplete and functional removal of self cells, while a part of the " bacterial mass"
pe
525 was found inside the cells of diseased oysters. Causes of lesions or death in infected
526 oysters are often the result of a combination of several factors including physical
527 movement of the Polydora itself and secondary cross infection due to multiple
528 pathogenic bacteria.
529
Acknowledgments
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530

531 This study was supported by the Professorial and Doctoral Scientific Research
532 Foundation of Huizhou University，the Team of Guangdong Provincial Department
tn

533 of Education：arine shellfish ecological breeding and disease prevention and control
534 innovation plan (2021kcxtd057).
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