Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses

Accepted Manuscript Experimental infection of Octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses. Vasileios Bakopoulos, Daniella White, Michail-Aggelos Valsamidis, Feli Vasilaki PII: DOI: Reference:

S0022-2011(17)30020-4 http://dx.doi.org/10.1016/j.jip.2017.01.008 YJIPA 6910

To appear in:

Journal of Invertebrate Pathology

Received Date: Revised Date: Accepted Date:

3 August 2016 12 January 2017 15 January 2017

Please cite this article as: Bakopoulos, V., White, D., Valsamidis, M-A., Vasilaki, F., Experimental infection of Octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses., Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip. 2017.01.008

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Title

2

Experimental infection of Octopus vulgaris (Cuvier, 1797) with Photobacterium

3

damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue

4

responses.

5 6

Authors

7

Vasileios, Bakopoulos*

8

Daniella, White

9

Michail-Aggelos, Valsamidis

10

Feli, Vasilaki

11 12

Department of Marine Sciences, School of the Environment, University of The Aegean,

13

University Hill, Mytilene 81100, Lesvos, Greece

14 15

*

16

University of The Aegean, University Hill, Mytilene 81100, Lesvos, Greece,, tel.:

17

++302251036870, email: [email protected]

Corresponding Author. Department of Marine Science, School of the Environment,

18 19

Abstract

20

Adult common octopus individuals were intramuscularly infected with Photobacterium

21

damsela subsp. piscicida in order to investigate if this species is sensitive to this

22

common and important fish pathogen. The fate of the bacterial antigens and the tissue

23

responses of Octopus vulgaris were studied employing immunohistochemical

24

techniques.

1

25

Strong reaction at the site of injection was evident from day 2 post-infection that

26

continued until day 14. Great numbers of hemocytes that were attracted at the site of

27

infection were involved in phagocytosis of bacteria. Very early in the infection, a

28

transition of cells to fibroblasts and an effort to isolate the infection was observed.

29

During the course of the study, very large necrotic cells were seen at the site of

30

infection, whereas during the later stages hemocytes with phagocytosed bacteria were

31

observed in well-defined pockets inside the muscle tissue. None of the internal organs

32

tested for the presence of the bacterium were positive with the exception of the digestive

33

gland where antigen staining was observed which was not associated with hemocyte

34

infiltration. The high doses of bacterial cells used in this experimental infection and the

35

lack of disease signs from Octopus vulgaris suggest that, under normal conditions,

36

octopus is resistant to Photobacterium damsela subsp. piscicida.

37 38

Keywords

39

Octopus vulgaris, tissue reaction, Photobacterium damsela subsp. piscicida,

40

immunohistochemistry

41 42 43

1. Introduction

44

Octopus vulgaris is a cephalopod species consumed traditionally especially in countries

45

bordering the Mediterranean Sea. According to FAO’s Globefish utility (2016), total

46

octopus (both Octopus maya and O. vulgaris) production for 2014 reached 370,000 t.

47

The main cephalopod consuming countries in the Mediterranean are Spain, Portugal,

48

Morocco, Mauritania, Greece and Italy (Baldrati, 1989). Since the second half of the

2

49

last century, and as a way of diversifying the fishing effort, O. vulgaris among other

50

cephalopods were considered as less conventional resources, and the capture of these

51

species was recommended (Pedrosa-Menabrito & Regenstein, 1988).

52

The need for diversification of aquaculture and for covering the demand for O. vulgaris

53

coupled by the increase of fishing costs and the depletion of stocks has led to efforts of

54

growing the species in captivity, with the leader in this effort to be Spain (Garcia &

55

Garcia, 2011). There are numerous reports investigating various aspects of octopus

56

culture in captivity (Iglesias, et al., 1997; Cagnetta & Sublimi, 2000; Iglesias, et al.,

57

2000; Iglesias, et al., 2002; Villanueva, et al., 2002; Carrasco, et al., 2003; Navarro &

58

Villanueva, 2003). However, O. vulgaris, despite these efforts, is still, in the majority of

59

cases, utilized in capture-based aquaculture, with the major obstacle for the completion

60

of the biological cycle in captivity to industrial standards / levels, being the paralarva

61

stage survival rate which is very low (Vaz-Pires, et al., 2004).

62

There are reports on diseases and pathogens affecting cephalopods in the wild and in

63

captivity. Cephalopods can be infected by a variety of agents including bacteria,

64

protozoa, metazoa, cestoda, trematoda, nematoda and crustacea. Octopus spp. infection

65

has been reported by Cytophaga-like and Pseudomonas species, in the mantle

66

(Castellanos-Martinez & Gestal, 2013 and references therein) and Vibrio lentus isolated

67

from the branchial heart caused lesions on the arms of the octopus (Farto, et al., 2003).

68

Additional reports (Hanlon, et al., 1984; Hanlon & Forsythe, 1990) have shown that

69

Octopus spp. can be infected externally in skin lesions by common Vibrio spp.,

70

Photobacterium damsela subsp. damsela, Aeromonas spp. present in the marine

71

environment. These bacterial species seem to be opportunistic pathogens since they can

72

be isolated from the external surfaces of wild or captive cephalopods (Ford, et al.,

3

73

1986). Protozoan infection of O. vulgaris has been reported by the coccidian Aggregata

74

octopiana (Gestal, et al., 1999a; Gestal et al. 2002; Mladineo & Jocic, 2005; Mayo-

75

Hernandez, et al., 2013) affecting the intestine and caecum. Metazoan parasites also

76

infect octopus, such as the cestode Phyllobothrium sp. and the nematode Cystidicola sp.

77

(Pascual, et al., 1996). The nematodes Anisakis sp., and Hysterothylacium sp. have been

78

found only in the lesser octopus (Eledone cirrhosa) (Gestal, et al., 1999b). Infections of

79

Pennella spp. (crustacean) have been reported to affect the condition of of

80

ommastrephid squids and thus, the parasite might cause a similar effect in O. vulgaris

81

which is also infected by the copepod Octopicola sp. (Pascual, et al., 1996; 1998).

82

O. vulgaris, like the rest of cephalopods, lacks a specific immune response and does not

83

possess immunological memory (Castellanos-Martinez & Gestal, 2013). Scientific

84

evidence suggests that O. vulgaris relies on innate immunity (non-adaptive) to defend

85

against infections, triggered by foreign antigens and implemented through the

86

mobilization of cells in the hemolymph, namely hemocytes and molecules dissolved in

87

the serum (opsonins, agglutinins, lysozyme) (Ford, 1992). Cephalopod hemocytes

88

contribute to the protection of the host against infection through phagocytosis,

89

encapsulation, infiltration or cytotoxic activities destroying or isolating pathogens

90

(Novoa, et al., 2002; Castellanos-Martinez, et al., 2014). Studies on the source,

91

morphology and function of O. vulgaris hemocytes suggest that these hemocytes are

92

produced in the white body organ located around the optic nerve (Cowden & Curtis,

93

1973; Bolognari, et al., 1980). Morphologically there seem to be two distinct cell

94

populations circulating in the hemolymph, large granulocytes with a U-shaped nucleus

95

containing basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm

96

and small granulocytes with a round nucleus occupying almost the entire cell and few or

4

97

not granules in the cytoplasm (Novoa, et al., 2002; Castellanos-Martinez, et al., 2014).

98

More recent studies (Troncone, et al., 2014) have identified yet another cell type the so-

99

called haemoblast-like cells which are smaller than the small granulocytes

100

(hyalinocytes) and large granulocytes and, in Sepia officinalis, tissue-adherent

101

hemocytes, or rhogocytes were found in the branchial heart complex (Beuerlein, et al.,

102

2002; Castillo, et al., 2015). Functionally, hemocytes infiltrate infected and/or damaged

103

tissue areas, phagocytize foreign and damaged material and produce oxygen and

104

nitrogen radicals (Rodriguez-Dominguez, et al., 2006; Castellanos-Martinez & Gestal,

105

2013; Gestal & Castellanos-Martinez, 2015). Finally, Castellanos-Martinez, et al.

106

(2014) showed that large granulocytes are the principal cells that develop phagocytosis

107

and ROS with small granulocytes exhibiting positive but small activities.

108

In view to the expanding aquaculture of the common octopus and to the reports that

109

indicate that common bacteria in sea water can infect wild or captive animals, the

110

objectives of this study were to investigate the sensitivity of experimentally infected O.

111

vulgaris to the common fish pathogen Photobacterium damsela subsp. piscicida, to

112

follow the fate of the pathogen upon entrance to the experimental animals and to

113

illustrate the tissue response by means of immuhistochemistry and light microscopy.

114 115

2. Materials & Methods

116

All chemicals used in this study were purchased from Sigma-Aldrich unless otherwise

117

stated.

118 119

2.1. Location and method of sampling

5

120

The octopuses (O. vulgaris) used in this study were caught from the wild in a coastal

121

location adjacent to the experimental facilities of the Department of Marine Sciences.

122

Collection lasted 14 days and all the 35 individuals collected had a body weight >500g

123

(body weight ranged from 676 to 877g) which is the requirement by the Greek

124

legislation. Octopuses were caught by free diving and by hand, being careful not to

125

cause any traumas. Individuals were placed in suitable buckets filled with seawater from

126

the collection area and covered with lid. Transportation to the experimental facilities did

127

not last more than 10min, since the distance from the collection area was less than 1km.

128 129

2.2. Aquaria, equipment and conditions

130

On arrival to the experimental facilities octopuses were immediately placed in plastic

131

aquaria filled with seawater from the collection area. Fourteen aquaria were used, 7 of

132

them holding 3 animals (infections) each, and another 7 holding two individuals

133

(controls) each. Each aquarium had a water holding capacity of 0.36m3 and was

134

separated in three or two compartments with custom-made wooden frames covered with

135

a net having a 0.5cm mesh size. Each four aquaria were connected with an EHEIM

136

Type 2217 pump which incorporates a canister with a solids, biological and activated

137

carbon filter and has a capacity of 1m3/h. Aeration of water was conducted with a

138

RESUN, ACO-003, electromagnetic pump having a capacity of 65 L/min and air was

139

distributed in each aquarium with airstones. Each aquarium had a lid to avoid the escape

140

of animals. Temperature throughout the experiment ranged between 16-18 οC and

141

dissolved oxygen was kept at 5.8-6.1mg/L. Temperature and oxygen were measured

142

daily with a WTW Oxi 315i probe, while pH and salinity were measured weekly and

143

ranged from 7.6-7.9 and 38,7-39.1‰, respectively. Total ammonia never exceeded

6

144

0.1mg/l (approximately 0.003mg/l of toxic unionized ammonia) (NH 3/NH4 test, SERA)

145

and nitrites never exceeded 0.2mg/l (NO 2 test, SERA). Every two days 1/3 of seawater

146

in the aquaria was renewed to avoid the accumulation of toxic metabolites.

147 148

2.3. Feeding

149

Octopuses were fed 7-8 fresh mussels or 2-3 rock crabs every 2 nd day, according to their

150

weight. Uneaten food and feces were removed twice a day.

151 152

2.4. Bacteria

153

The common marine fish pathogen Photobacterium damsela subsp. piscicida (Phdp,

154

hereafter) (Magarinos, et al. 1994a; Bakopoulos, et al., 1997a; Le Breton, 1999; 2009;

155

Athanassopoulou & Bitchava, 2010; Bakopoulos, et al., 2015) was used throughout the

156

study.

157

A colony of the pathogen was placed in BHIB (HIMEDIA) with 2% NaCl and left to

158

grow for 48h at 22οC. The culture was then centrifuged (LabNet, Hermle Z200A) to

159

isolate bacterial cells at 1,750g for 1h at 4 οC. Bacterial cells were resuspended in sterile

160

2% NaCl and at an optical density of 1 at 605nm (Merck, spectroquant Nova 60,

161

photometer) corresponding to 5×109 bacterial cells/ml, as it was previously determined

162

using the plate count method.

163 164

2.5. Experimental design and sampling

165

Collected octopuses were allowed to acclimatize in captivity for at least 1 week, since

166

the last collected animals. Twenty one octopuses were infected intramuscularly with

167

100μl of the bacterial suspension prepared as above, after sedation using a solution of

7

168

2.5% ethanol in seawater (Rodriguez-Dominguez, et al., 2006; Gleadall, 2013;

169

Andrews, et al., 2013), while another fourteen individuals were injected with the same

170

volume of sterile 2% NaCl, serving as controls. The location of injection for each

171

octopus was 4cm from the tip of the relaxed 5th arm. The point of infection was sampled

172

every 2 days post-infection from a different individual. In total, 21 samples (1 sample

173

per individual from 3 individuals per sampling date) of the infected tissue were

174

collected (days 2, 4, 6, 8, 10, 12 & 14 post-infection) and another fourteen samples of

175

muscle tissue were collected at the same days post-infection from the controls for

176

comparison purposes. Infected and control octopuses that were used for the collection of

177

the day 14 samples were sacrificed with an overdose of ethanol (20%) in order to

178

sample the internal organs (gills, digestive gland & kidney). Tissue samples were

179

immediately placed in 10% phosphate buffered formalin.

180

After the end of the experiments all the remaining animals were kept for a period of 21

181

days in order to evaluate the development of any disease signs and behavior. Since no

182

disease was observed and behavior was normal (assessed by activity, body coloration,

183

feeding activity) (for a description of the behavior of healthy Octopus vulgaris in

184

captivity see for example Boycott, 1954; Hochner, 2008) the remaining animals were

185

released back to the wild.

186 187

2.6. Production of sea bass anti-Phdp polyclonal serum

188

Hyperimmune sea bass (Dicentrarchus labrax, L.) polyclonal serum was prepared

189

according to the method of Bakopoulos, et al. (1997b). Briefly, the bacterial culture

190

prepared as described previously was formalin-inactivated (3% v/v) for 24h at 4 οC.

191

Bacterial cells were then washed twice in sterile 2% NaCl. Supernatants of the culture

8

192

containing extracellular products (ECPS) were incubated, after the addition a 15%

193

solution of sodium metabisulphite (10 ml/L) for the neutralization of formalin,

194

overnight at room temperature. Inactivated bacterial cells were resuspended in the

195

neutralized ECPs solution at 5×109 bacterial cells/ml. Sea bass weighing 15g were

196

intraperitoneally injected with 200μl of the suspension thrice every 25 days and were

197

exsanguinated 20 days from the last immunization. Blood was allowed to clot for 10min

198

at r.t. and overnight at 4οC. Samples were centrifuged for 10min at 2,500g for the

199

isolation of serum. Sera were then pooled, mixed well, aliquoted and stored at -85 οC for

200

future use. Fish handling was performed under sedation using 0.5%, 2-phenoxyethanol.

201 202

2.7. Histology

203

Collected tissue samples remained at least for 48h in the formalin solution and were

204

then embedded in paraffin. Three sequential sections (25μm apart from each other) 5μm

205

thick from each sample were cut using a LEICA RM2125 RTS microtome and were

206

placed in sensitized glass slides (Superfrost plus, Thermo Scientific). Samples were

207

utilized in immunohistochemistry and counterstained with Harry’s hematoxylin (Drury

208

& Wallington, 1980).

209 210

2.8. Immunohistochemistry (IHC)

211

Tissue sections of each sample were used in IHC utilizing the method of Adams &

212

Marin de Mateo (1994) with modifications. Briefly, tissue sections were incubated with

213

sea bass anti-Phdp hyperimmune polyclonal serum, diluted 1:50, and overnight at 4 οC.

214

The serum was removed, slides washed and mouse anti-sea bass IgM monoclonal

215

antibody (Bakopoulos, et al., 1997b) was added and samples were incubated for 2h at

9

216

r.t. After removal and washing the slides, goat anti-mouse IgG-HRP was added for

217

30min at r.t. and after removal the reaction was visualized using diaminobenzidine

218

(DAB). Samples were then counterstained with Harry’s hematoxylin allowed to dry and

219

inspected under microscope (OLYMPUS CH20). Samples from non-infected tissues

220

were utilized, serving as negative controls and further negative controls for the assay

221

(devoid of the step with sea bass anti-Phdp hyperimmune polyclonal serum incubation)

222

using infected tissue samples, were utilized.

223 224

3. Results

225

Experimental animals did not show evidence of disease development or discomfort

226

during the experimental period as assessed by observation of their overall (Andrews et

227

al., 2013) and feeding activity. During the first 1-2h of the injection, animals restrained

228

the use of the arm that was infected beyond the point of injection and towards the edge

229

of the arm.

230 231

3.1. Microscopy of control tissue samples

232 233

The normal histological structure of unharmed octopus muscle is shown in Figure 1A.

234

Note the absence of hemocytes between the undisturbed muscle fibers. Figure 1B

235

illustrates the effect of injection on the architecture of muscle tissue minutes post-

236

injection (pi). Muscle samples from sham-injected individuals that were sampled at day

237

2 pi (Figure 1C), except of the disturbance of the muscle tissue, only a handful of

238

hemocytes are observed (arrows) at the location of injection. On day 4 pi (Figure 1D),

10

239

hemocytes (arrows) are observed present and migrating through the muscle tissue

240

towards the point of injection.

241 242

Insert Figure 1

243 244

The microscopic picture of the muscle tissue at the point of injection on day 6 pi (Figure

245

2A) is similar as described before with only a few hemocytes (arrows) being observed.

246

Samples from days 8 (Figure 2B) and 10 (Figure 2C) pi showed a noticeable attraction

247

of hemocytes (arrows) at the point of injection. Note the presence of hemocytes with

248

dendritic pseudopodia (arrows). During days 12 (Figure 2D) and 14 (data not shown) pi,

249

while the microscopic picture at the point of injection remained unchanged, large

250

numbers of hemocytes (arrows) were observed migrating towards the point of injection

251

through the adjacent undisturbed muscle tissue. As it is evident, none of the control

252

samples reacted with any of the probes and reagents used in IHC.

253 254

Insert Figure 2

255 256

3.2. Microscopy of infected specimens’ tissue samples

257 258

At day 2 pi, an intense attraction of hemocytes at the site of injection was observed

259

(Figure 3A). The tissue reactions were either diffuse or well defined. Phagocytosis of

260

bacteria (arrows) was evident from large hemocytes, recognized by the high ratio of

261

cytoplasm to nucleus and by their U-shaped nucleus (i.e. top arrow, Figure 3A). To the

262

left, a group of cells had phagocytosed foreign antigen (asterisks, Figure 3A) and they

11

263

were in various stages of degeneration evident as cell swelling and in some cases

264

disappearance of the nucleus. This tissue reaction seems to be in the first stages of

265

encapsulation as noted by nucleated elongated cells (resembling fibroblasts) developing

266

around the reaction site (arrowheads, Figure 3A). Figure 3B, also from the 2nd day pi,

267

shows detail of severe infiltration of hemocytes at the point of injection in muscle

268

tissue. As it is evident antigens are phagocytosed throughout the infiltrated area from

269

hemocytes in the presence of many more which did not stain for antigens of the

270

pathogen. Evidence of cells transforming at the edges of the site of infection to

271

elongated forms (fibroblasts) in order to isolate the infection was noted (Figure 3B).

272

On day 4 pi (Figures 3C and 3D) there have been similar findings as for day 2 pi. Figure

273

3C shows an overview of the infected muscle tissue. A borderline of the degenerated

274

muscle tissue is formed by attracted hemocytes. Two zones were noted. A more defined

275

border made up of hemocytes with phagocytosed antigens (arrowheads to the left,

276

Figure 3C), followed by degenerated tissue and then another zone with hemocyte

277

infiltration defining the area where the infectious agent is located (arrowheads to the

278

right, Figure 3C). As seen in Figure 3D, antigen was restricted into certain areas

279

(arrows) in the infected tissue surrounded by hemocytes. On day 6 similar

280

histopathological signs were observed as in day 4. Figure 3E shows detail of the

281

isolation of foreign antigens in the muscle and reveals strong phagocytosis of antigens

282

(brown staining) and flattening of the cell nucleus of hemocytes forming fibroblasts.

283 284

Insert Figure 3

285 286

On day 8 necrotic areas in the muscle were seen in higher proportion (Figure 4A).

12

287

The muscle tissue had lost its structure and enlarged necrotic cells fill the area. These

288

cells are observed adjacent to muscle tissue containing normal hemocytes (arrows,

289

Figure 4A) but there was no antigen staining. The microscopic view of samples at day

290

10 pi was similar to days 6 and 8 pi (data not shown). Infected muscle sampled on day

291

12 (Figures 4B and 4C), revealed persistence of the tissue reaction with hemocyte

292

infiltration and phagocytosis of antigens (arrowhead, Figure 4B). Next to these diffuse

293

reaction areas there were hemocytes carrying bacterial antigens isolated from the rest of

294

the muscle tissue (arrows, Figure 4B). The foreign antigen, phagocytosed, was isolated

295

in pockets (Figure 4C) inside the muscle tissue. On day 14 (Figure 4D), less bacterial

296

antigen was seen in the infected area and the overall impression was that the strong

297

infiltration of hemocytes is weakening. A stronger evidence of fibrolasts around the

298

infection was observed (arrowheads, Figure 4D).

299 300

Insert Figure 4

301 302

3.3. Microscopy of internal organs samples

303 304

Insert Figure 5

305 306

No staining for antigens was observed in gill samples from both control and infected

307

specimens (Figure 5A & 5B, respectively). No staining for antigens was observed in

308

samples of digestive gland collected from control specimens (Figure 5C). Only a small

309

number of hemocytes could be identified (arrowheads, Figure 5C). In contrast, antigen

310

staining was evident in the digestive gland of infected specimens in certain areas and in

13

311

a dispersed form at the margins of the cords of digestive gland cells (Figure 5D,

312

arrows). Interestingly, only a few hemocytes were observed (arrowheads, Figure 5D)

313

not always stained for antigens. Kidney samples from both control and infected

314

specimens were devoid of bacterial antigens (Figure 5E & 5F, respectively).

315 316

4. Discussion

317 318

4.1. O. vulgaris sensitivity to Phdp

319 320

Phdp, an important marine fish pathogen (Bakopoulos, et al., 1997a), did not caused

321

disease to O. vulgaris, after intramuscular injection and in the short 14-day-long present

322

study. The infectious doses used have been extremely high (5×10 9 bacterial cells/ml)

323

according to previous studies on fish and this is in favor to the notion of a certain

324

resistance of octopus to this pathogen. Indeed, a bacterial dose of 1.5×10 5 cells/ml

325

caused 53-98% mortality in sea bass (Bakopoulos, et al., 2003a); whereas 1.75×10 5 and

326

3.25×104 caused 90% and 45% mortality, respectively, in the same species

327

(Bakopoulos, et al., 2015). In both these studies, the plateau of mortalities was reached

328

within a week post-infection. This is a positive result since this pathogen being endemic

329

and causing disease to cultured sea bass or sea bream (Spatus aurata, L.) may not affect

330

octopus reared in areas that are used for the culture of these fish species. This

331

suggestion is strengthened by the fact that, although the closely related bacterium Ph.

332

damsela subsp. damsela has been isolated from external surfaces and wounds of

333

octopus (Hanlon, et al., 1984; Farto, et al., 2003), there is no report so far of

334

photobacteriosis caused by either bacteria in reared O. vulgaris. Nevertheless, additional

14

335

tests are required employing immersion infections for longer periods and with

336

specimens with external lesions to consider these conditions of infection as well.

337 338

4.2. Tissue responses of O. vulgaris injected with sterile 2% NaCl (controls)

339

Sham injection in the control animals neither produced an external wound nor an

340

intense attraction of hemocytes (as in an inflammatory response) at the point of

341

injection. Indeed, all the samples collected from day 0 to day 6 showed only a

342

disturbance of the normal muscle structure at the point of injection and only a handful

343

of hemocytes. Noticeable attraction of hemocytes was observed from day 8 onwards

344

and until the end of the experiment. Worth noting are the changes observed in

345

hemocytes located outside the hemolymph vessels which included the elongation of

346

their nuclei and cytoplasm and the creation of dendritic pseudopodia in the areas of

347

damaged muscle tissue. This is in agreement to observations during wound healing in

348

cuttlefish (Feral, 1988) and in the photodocumentation of hemocytes by Castellanos-

349

Martinez, et al. (2014) and Troncone, et al. (2014), in vitro.

350

There are numerous reports regarding tissue responses of various cephalopod species

351

during wound healing (Feral, 1988; Polgase, et al., 1983; Bullock, et al., 1987; Pascual,

352

et al., 2006). These sequentially include some closure of the wound through muscle

353

contraction and epidermal curvature, the attraction of hemocytes within the first 24h of

354

trauma which transform from round-ovoid cells to fusiform cells when leave blood

355

vessels, the formation of a cellular (hemocytes) dermal plug, the progressive

356

organization of migrated hemocytes and their transformation to fibroblasts and a return

357

of epidermal morphology to normal state at the point of the lesion from 14 days

358

onwards. These steps in healing were not observed in the control specimen samples

15

359

collected in this study, apart from the later attraction of hemocytes at the point of

360

injection and their transformation when present outside vessels. This is probably due to

361

that a single injection through the skin differed by far from an open wound, as was the

362

case for the aforementioned reports.

363 364

4.3. Muscle responses of O. vulgaris injected with Phdp

365 366

The reaction of the tissue at the location of injection was very quick and strong, diffuse

367

or well defined, as it is evident from the samples collected at day 2 post-infection.

368

Phagocytosis of bacteria was evident from large hemocytes (Figure 3A). These

369

observations are in agreement with the studies of Castellanos-Martinez, et al. (2014) on

370

the functionality of hemocytes of octopus in vitro. As soon as 48h post-introduction of

371

bacteria there is evidence of isolation of the infection through the creation of a capsule

372

around necrotic cells and foreign antigens in the form of nucleated elongated cells

373

(resembling fibroblasts). A similar early transformation was noted also in earlier studies

374

by Polglase, et al. (1983), Bullock, et al. (1987) and Feral (1988). By day 4 post-

375

infection the reaction becomes more organized with hemocytes creating borderlines

376

between normal and damaged, infiltrated with bacteria, tissue. This “double tier” was

377

also noted by Bullock, et al. (1987) although in their study this or even a third tier was

378

evident up to 36 h.p.i. On days 4, 6 & 8 the reactions were similar but the proportion of

379

necrotic cells without any foreign antigen present increased. These necrotic cells were

380

very large in comparison to normal hemocyte size (Figure 4A) with both cytoplasm and

381

nucleus being swollen. Since no bacteria staining was observed, this necrosis may be

16

382

the result of tissue reactions to damage and our explanation is in agreement with a

383

similar suggestion by Bullock, et al. (1987).

384

In days 10, 12 and 14, the infiltration by hemocytes at the location of infection is still

385

strong but the proportion of necrotic cells is reduced and the hemocytes involved in

386

clearing the infected tissue seem to be in a better state. Throughout the study, even from

387

the 2nd day, hemocytes acted towards isolating the infection and necrotic areas through

388

transformation to fibroblast cells evidenced as flattening of the nucleus and of the

389

cytoplasm. The last days of the experiment the foreign antigen was enclosed in pockets

390

surrounded by healthy muscle tissue. These observations are in agreement to the

391

findings of Rodriguez-Dominguez, et al. (2006) and Castellanos-Martinez & Gestal

392

(2013).

393

Hemocytes in this study were actively and intensely involved in the phagocytosis of the

394

pathogen as it was documented by the specific IHC staining of the samples. This in vivo

395

study specifically demonstrated foreign antigens phagocytosis by hemocytes and it is in

396

agreement with previous studies on cephalopods describing the involvement of

397

clearance of foreign antigens by hemocytes in vivo or in vitro (Malham, et al., 1997;

398

Rodriguez-Dominguez, et al., 2006; Castellanos-Martinez & Gestal, 2013).

399 400

4.4. Gills, kidney and digestive gland responses of O. vulgaris injected with Phdp

401 402

Gills in this study did not stained for the presence of antigens of Phdp. This is in

403

contrast to the study of Bayne (1973) who suggested that gill tissue, and not circulating

404

hemocytes, was the primary site for clearing the bacterium Serratia marcescens from

405

the circulatory system in Octopus dofleini. The reason might be that infection in our

17

406

study was performed intramuscularly and not intravenously. Similarly, no evidence of

407

bacterial antigens presence was noted in kidney samples in this study. In contrast,

408

Sangster & Smolovitz (2003) describing V. alginolyticus infection of cultured cuttlefish,

409

reported the presence of the pathogen in both the kidney and gills. Differences in the

410

hemocyte types described for cuttlefish (Le Pabic, et al., 2014) in comparison to

411

octopus (Troncone, et al., 2014), may influence aspects of the cellular immune response

412

(i.e. organ location of phagocytosed antigens) in different cephalopods. In addition, the

413

cases report of Sangster & Smolovitz (2003) concerned animals held in captivity and in

414

contact with pathogenic bacteria for an undefined period of time, which presumably

415

were infected through lesions of the integument. A much longer period of contact with

416

V. alginolyticus is also suggested by the mature granulomatous lesions described in the

417

reproductive organs of these species. Both, hemocyte, route and period of infection as

418

well as pathogen differences may account for the differences of findings reported in this

419

study.

420

A novel finding of this study is that antigen staining was observed in tissue sections of

421

the digestive gland of infected octopus. The presence of antigen was not linked to a

422

proportionate presence (infiltration) of hemocytes or the presence of bacterial colonies

423

(Figure 5D). This may suggest that staining concerned soluble antigens of the pathogen

424

such as toxic extracellular products (Magarinos, et al., 1994b; Bakopoulos, et al.,

425

2003b) which cannot be visualized in histochemistry but rather in IHC with specific

426

immunological probes. To our knowledge, this is the first report of the involvement of

427

the digestive gland in foreign antigens clearance, since various studies on infections of

428

cephalopods with various bacteria did not reported their presence microscopically in the

429

digestive gland (Bayne, 1973; Bullock, et al., 1987; Ford, 1992; Sangster & Smolovitz,

18

430

2003; Castellanos-Martinez & Gestal, 2013; Castellanos-Martinez, et al., 2014; Castillo,

431

et al., 2015). In contrast, the digestive gland has been pinpointed as the organ of

432

accumulation and clearance of heavy metals in cephalopods (Bustamante, et al., 2002;

433

2006) and paralytic shellfish toxins (Monteiro & Costa, 2011; Lopes, et al., 2014) but

434

also, the digestive gland seems to have a key role in clearance of infectious substances

435

in the common octopus.

436

4.5. Conclusions

437

Extremely high doses of the marine fish pathogen Photobacterium damsela subsp.

438

piscicida delivered by intramuscular injection did not caused disease to the common

439

octopus. In contrast, the pathogen provoked a rapid and very intense inflammatory

440

response at the point of injection. Great numbers of hemocytes that were attracted at the

441

point of infection, phagocytosed the pathogens, as it was illustrated by IHC, and

442

restricted the infection by transforming to fibroblasts at the periphery of infected areas.

443

In contrast, in controls, a more delayed and less intense attraction of hemocytes at the

444

point of injection was observed. The gills and kidney of infected specimens, in contrast

445

to studies on other cephalopods, did not stained for the presence of antigens. Such

446

staining of antigens was observed, however, in the digestive gland of infected

447

specimens, without the presence of corresponding numbers of bacterial colonies or

448

hemocytes with phagocytosed antigens. These findings suggest reaction of the

449

antibacterial serum with soluble antigens of the bacterium and that the digestive gland

450

may be involved in foreign material clearance.

451 452

5. Aknowledgements

19

453

This study was funded by institutional funds.

454 455

6. Ethics

456

The work presented in the article has been carried out in an ethical way and according to

457

the EU Directive 2010/63/EU for animal experiments. Specifically, the whole

458

experimental procedure (specimens used, animal maintenance, experimental protocols

459

and animal release) was approved by the Committee for the Assessment of Protocols,

460

according to article 37 of the Presidential Decree 56/2013 conforming to article 36, 2nd

461

paragraph and article 38 of EU Dir 2010/63/EE (approval No.: 1108/07-11-2016). This

462

approval and the experimental protocol are attached as supplemental material to this

463

article.

464 465

7. References

466

Adams, A., Marin de Mateo, M., 1994. Immunohistochemical detection of fish

467

pathogens. In: Stolen, J.S., Fletcher, T.C., Kaatari, S.L., Rawley, A.F. (Eds),

468

Techniques in Fish Immunology 3. SOS Publications, Fair Heaven, New Jersey, U.S.A.,

469

pp. 133-144.

470 471

Andrews, P.L.R., Darmaillacq, A-S., Dennison, N., Gleadall, I.G., Hawkins, P.,

472

Messenger, J.B., Osorio, D., Smith, V.J., Smith J.A., 2013. The identification and

473

management of pain, suffering and distress in cephalopods, including anaesthesia,

474

analgesia and humane killing. J. Exp. Mar. Biol. Ecol. 447, 46-64.

475

20

476

Athanassopoulou, F., Bitchava, K., 2010. Main pathological conditions in

477

Mediterranean marine finfish culture. In: Koumoundouros G. (Ed.), Recent Advances

478

in Aquaculture. Transworld Research Network, Kerala, India, pp. 149-201.

479 480

Bakopoulos V., Nikolaou I., Kalovyrna N., Amirali E., Kokkoris G., Spinos E., 2015.

481

Prevention of fish photobacteriosis. Comparison of the efficacy of intraperitoneally

482

administered commercial and experimental vaccines. Mediterranean Mar. Sci. 16(2),

483

385-392.

484 485

Bakopoulos, V., Pearson, M., Volpatti, D., Gousmani, L., Adams, A., Galeotti, M.,

486

Dimitriadis, G.J., 2003b. Investigation of media formulations promoting differential

487

antigen expression by Photobacterium damsela subsp. piscicida and recognition by sea

488

bass, Dicentrarchus labrax, (L.), immune sera. J. Fish Dis. 26(1), 1-13.

489 490

Bakopoulos, V., Peric, Z., Rodger, H., Adams, A., Richards, R.H., 1997a. First report of

491

fish Pasteurellosis from Malta. J. Aquat. Anim. Health 9, 26-33.

492 493

Bakopoulos, V., Volpatti, D., Adams, A., Galleotti, M., Richards, R.H., 1997b.

494

Qualitative differences in the immunological response of rabbit, mouse and sea bass,

495

Dicentrarchus labrax, L, to the injection of Pasteurella piscicida, the causative agent

496

of fish Pasteurellosis. Fish Shellfish Immunol. 7, 161-174.

497 498

Bakopoulos,V., Volpatti, D., Gusmani, L., Galeotti, M., Adams, A., Dimitriadis G.J.,

499

2003a. Vaccination trials of sea bass, Dicentrarchus labrax (L.), against

21

500

Photobacterium damsela subsp. piscicida, using novel vaccine mixtures. J. Fish Dis.

501

26 (2), 77-90.

502 503

Baldrati, G., 1989. Handling, marketing and processing of cephalopods in Italy.

504

Industria Cons. 64, 353– 355.

505 506

Bayne, C.J., 1973. Internal defense mechanisms of Octopus dofleini. Malacol. Rev. 6,

507

13-17.

508 509

Beuerlein, K., Lohr, S., Westermann, B., Ruth, P., Schipp, R., 2002. Components of the

510

cellular defense and detoxification system of the common cuttlefish Sepia officinalis

511

(Mollusca, Cephalopoda), Tissue Cell 34(6), 390-396.

512 513

Bolognari, A., Fasulo, S., Licata, A., 1980. A preliminary comparison of the leukocyte

514

series of the cephalopod Todannles saginams and the granulocyte series in the bone

515

marrow of the rat Rattus rattus. Bull. Zool. 41, 221-225.

516 517

Boycott, B.B., 1954. Learning in Octopus vulgaris and other cephalopods.

518

Pubblicazioni della Stazione Zoologica di Napoli 25, 67–93.

519 520

Bullock, A.M., Polglase, J.L., Phillips, S.E., 1987. The wound healing and haemocyte

521

response in the skin of the lesser octopus Eledone cirrhosa (Mollusca: Cephalopoda) in

522

the presence of Vibrio tubiashii. J. Zool. London 211, 373-385.

523

22

524

Bustamante, P., Lahaye, V., Durnez, C., Churlaud, C., Caurant, F., 2006. Total and

525

organic Hg concentrations in cephalopods from the North East Atlantic waters:

526

influence of geographical origin and feeding ecology. Sci. Total Environ. 368, 585-596.

527 528

Bustamante, P., Teyssie, J.L., Fowler, S.W., Cotret, O., Danis, B., Miramand, P.,

529

Warnau, M., 2002. Biokinetics of zinc and cadmium accumulation and depuration at

530

different stages in the life cycle of the cuttlefish Sepia officinalis. Mar. Ecol. Prog. Ser.

531

231, 167-177.

532 533

Cagnetta, P., Sublimi, A., 2000. Productive performance of the common octopus

534

(Octopus vulgaris C.) when fed on a monodiet. Recent Advances in Mediterranean

535

Aquaculture Finfish Species Diversification. Cahiers Options Mediterraneennes 47,

536

331–336.

537 538

Castellanos-Martinez, S., Gestal, C., 2013. Pathogens and immune response of

539

cephalopods. J. Exp. Mar. Biol. Ecol. 447, 14-22.

540 541

Castellanos-Martinez, S., Prado-Alvarez, M., Lobo-da-Cunha, A., Azevedo, C., Gestal,

542

C., 2014. Morphologic, cytometric and functional characterization of the common

543

octopus (Octopus vulgaris) hemocytes. Develop. Comp. Immunol. 44, 50-58.

544 545

Castillo, M.G., Salazar, K.A., Joffe, N.R., 2015. The immune response of cephalopods

546

from head to foot. Fish Shell. Immunol. 46, 145-160.

547

23

548

Carrasco, J.F., Rodrıguez, C., Rodrıguez, M., 2003. Cultivo intensivo de paralarvas de

549

pulpo (Octopus vulgaris, Cuvier 1797) utilizando como base de la alimentacion zoeas

550

vivas de crustaceos. IX Congreso Nacional de Acuicultura, Cadiz, 12–16 Mayo 2003.

551 552

Cowden, R.R., Curtis, S.K., 1973. Observations on living cells dissociated from

553

leukopoietic organ of Octopus briareus. Exp. Mol. Pathol. 19, 178–185.

554 555

Drury, R.A.B., Wallington, E.A., 1980. Carleton’s histological techniques. 5th Edition.

556

Oxford University Press, Oxford, U.K.

557 558

Farto, R., Armada, S.P., Montes, M., Guisande, J.A., Perez, M.J., Nieto, T.P., 2003.

559

Vibrio lentus associated with diseased wild octopus (Octopus vulgaris). J. Invertebr.

560

Pathol. 83, 149–156.

561 562

FAO Globefish utility, 2016. http://www.globefish.org/octopus-june-2016.html

563

(accessed 11/06/2016)

564 565

Feral J-P., 1988. Wound healing after arm amputation in Sepia officinalis

566

(Cephalopoda: Sepioidea). J. Invertebr. Pathol. 52, 380-388.

567 568

Ford, L.A., 1992. Host defense mechanisms of cephalopods. Annual Rev. Fish Dis. 2,

569

25–41.

570

24

571

Ford, L.A., Alexander, S.K., Cooper, K.M., Hanlon, R.T., 1986. Bacterial populations

572

of normal and ulcerated mantle tissue of the squid, Lolliguncula brevis. J. Invertebr.

573

Pathol. 48, 13–26.

574 575

Garcia, J.G., Garcia, B.G., 2011. Econometric model of viability/profitability of octopus

576

(Octopus vulgaris) ongrowing in sea cages. Aquac. Intl. 19, 1177–1191.

577 578

Gestal, C., Abollo, E., Pascual, S., 2002. Observations on associated histopathology

579

with Aggregata octopiana infection (Protista: Apicomplexa) in Octopus vulgaris. Dis.

580

Aquat. Org. 50, 45–49.

581 582

Gestal, C., Belcari, P., Abollo, E., Pascual, S., 1999b. Parasites of cephalopods in the

583

northern Tyrrhenian Sea (western Mediterranean): new host records and host

584

specificity. Scientia Marina, 63(1), 39-43.

585 586

Gestal, C., Castellanos-Martinez, S., 2015. Understanding the cephalopod immune

587

system based on functional and molecular evidence. Fish Shell. Immunol. 46, 120-130.

588 589

Gestal, C., Pascual, S., Corral, L., Azevedo, C., 1999a. Ultrastructural aspects of the

590

sporogony of Aggregata octopiana (Apicomplexa, Aggregatidae), a coccidian parasite

591

of Octopus vulgaris (Mollusca, Cephalopoda) from NE Atlantic coast. Europ. J.

592

Protistol. 35, 417–425.

593

25

594

Gleadall, I.G., 2013. The effects of prospective anaesthetic substances on cephalopods:

595

Summary of original data and a brief review of studies over the last two decades. J. Exp.

596

Mar. Biol. Ecol. 447, 23-30.

597 598

Hanlon, R.T., Forsythe, J.W., 1990. Diseases of molluscs: Cephalopoda, In: Kinne, O.,

599

(Ed). Diseases of marine animals, vol. 3. Cephalopoda to urochordata. Hamburg.

600

Germany: Biologische. Anstalt. Helgokmd, pp. 23-16.

601 602

Hanlon, R.T., Forsythe, J.W., Cooper, K.M., Dinuzzo, A.R., Folse, D.S., Kelly, M.T.,

603

1984. Fatal penetrating skin ulcers in laboratory-reared octopuses. J. Invertebr. Pathol.

604

44, 67–83.

605 606

Hochner, B., 2008. Octopuses. Curr. Biol. 18, 897–898.

607 608

Iglesias, J., Otero, J.J., Moxica, C., Fuentes, L., Sanches, F.J., 2002. Paralarvae culture

609

of octopus (Octopus vulgaris) using artemia and zoeas, and first data on juvenile growth

610

up to eight months of age. Seafarming Today and Tomorrow-Extended Abstracts and

611

Short Communications. Presented at the Aquaculture Europe 2002, Trieste, Italy,

612

October 16– 19. EAS Special Publication, vol. 32. European Aquaculture Society, EAS,

613

Oostende, Belgium, pp. 268–269.

614 615

Iglesias, J., Sanchez, F.J., Otero, J.J., 1997. Primeras experiencias sobre el cultivo

616

integral del pulpo (Octopus vulgaris Cuvier) en el Instituto Espanol de Oceanografıa.

617

Actas del VI Congreso Nacional de Acuicultura. Cartagena, Spain, 9, 10 and 11 July.

26

618 619

Iglesias, J., Sanchez, F.J., Otero, J.J., Moxica, C., 2000. Culture of octopus (Octopus

620

vulgaris, Cuvier): present knowledge, problems and perspectives. Recent advances in

621

Mediterranean Aquaculture Finfish Species Diversification. Cahiers Options

622

Mediterraneennes 47, 313– 322.

623 624

Le Breton, A.D., 1999. Mediterranean finfish pathologies: present status and new

625

developments in prophylactic methods. Bull. Eur. Ass. Fish Pathol. 19, 250-253.

626 627

Le Breton, A.D., 2009. Vaccines in Mediterranean aquaculture: Practice and needs, In:

628

Rodgers, C., Basurco, B. (Eds). The use of veterinary drugs and vaccines in

629

mediterranean aquaculture. Options Méditerranéennes, Series A, No. 86, CIHEAM,

630

Zaragosa, Spain, pp. 147-154.

631 632

Le Pabic, C., Goux, D., Guillamin, M., Safi, G., Lebel J-M., Koueta, N., Serpentini A.,

633

2014. Hemocyte morphology and phagocytic activity in the common cuttlefish (Sepia

634

officinalis). Fish Shell. Immunol. 40, 362-373.

635 636

Lopes, V.M., Baptista, M., Repolho, T., Rosa, R., Costa, P.R., 2014. Uptake, transfer

637

and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus

638

vulgaris). Aquat. Toxicol. 146, 205– 211.

639

27

640

Magarinos B., Romalde J.L., Lemos M.L., Barja J.L. & Toranzo A.E., 1994b. Iron

641

uptake by Pasteurella piscicida and its role in pathogenicity for fish. App. Env.

642

Microbiol. 60, 2990–2998.

643 644

Magarinos B., Romalde J.L., Santos Y., Casal J.F., Barja J.L. & Toranzo A.E., 1994a.

645

Vaccination trials on gilthead sea bream (Sparus aurata) against Pasteurella piscicida.

646

Aquac. 120, 201–208.

647 648

Malham, S.K., Runham, N.W., Secombes C.J., 1997. Phagocytosis by haemocytes from

649

the Lesser Octopus Eledone cirrhosa. Iberus 15(2), 1-11.

650 651

Mayo-Hernández, E., Barcala, E., Berriatua, E., García-Ayala, A., Muñoz, P., 2013.

652

Aggregata (Protozoa: Apicomplexa) infection in the common octopus Octopus vulgaris

653

from the West Mediterranean Sea: The infection rates and possible effect of faunistic,

654

environmental and ecological factors. J. Sea Res. 83, 195-201.

655 656

Mladineo, I., Jozić, M., 2005. Aggregata infection in the common octopus, Octopus

657

vulgaris (Linnaeus, 1758), Cephalopoda: Octopodidae, reared in a flow-through system.

658

Acta Adriatica 46(2), 193-199.

659 660

Monteiro, A., Costa P.R., 2011. Distribution and selective elimination of paralytic

661

shellfish toxins in different tissues of Octopus vulgaris. Harmful Algae 10, 732-737.

662

28

663

Navarro, J.C., Villanueva, R., 2003. The fatty acid composition of Octopus vulgaris

664

paralarvae reared with live and inert food: deviation from their natural fatty acid profile.

665

Aquac. 219, 613–631.

666 667

Novoa, B., Tafalla, C., Guerra, A., Figueras, A., 2002. Cellular immunological

668

parameters of the octopus, Octopus vulgaris. J. Shell. Res. 21, 243–248.

669 670

Pascual, S., Gestal, C., Estevez, J.M., Rodriguez, H., Soto, M., Abollo, E., Arias, C.,

671

1996. Parasites in commercially-exploited cephalopods (Mollusca, Cephalopoda) in

672

Spain: an updated perspective. Aquac. 142, 1–10.

673 674

Pascual, S., Rocha, F., Guerra, A., 2006. Gross lesions in the Hubb octopus Octopus

675

hubbsorum. Mar. Biol. Res. 2, 420–423.

676 677

Pascual, S., Gonzalez A.F., Guerra, A., 1998. Effect of parasitism on the productivity of

678

the ommastrephid stocks in Galician waters (NW Spain): economic loss. Iberus, 16(2),

679

95-98.

680 681

Pedrosa-Menabrito, A., Regenstein, J.M., 1988. Shelf-life extension of fresh fish—a

682

review: part I. Spoilage of fish. J. Food Qual. 11, 117– 127

683 684

Polglase, J.L., Bullock, A.M., Roberts, R.J., 1983. Wound healing and the haemocyte

685

response in the skin of the lesser octopus, Eledone cirrhosa (Mollusca: Cephalopode). J.

686

Zool. Lond. 201, 185-204.

29

687 688

Rodriguez-Dominguez, H., Soto-Bua, M., Iglesias-Blanco, R., Crespo-Gonzalez, C.,

689

Arias Fernandez, C., Garcia-Estevez, J., 2006. Preliminary study on the phagocytic

690

ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro.

691

Aquac. 254, 563–570.

692 693

Sangster, C.R., Smolovitz, R.M., 2003. Description of Vibrio alginolyticus Infection in

694

Cultured Sepia officinalis, Sepia apama, and Sepia pharaonic. Biol. Bull. 205, 233-234.

695 696

Troncone, L., De Lisa, E., Bertapelle, C., Porcellini, A., Laccetti, P., Polese, G., Di

697

Cosmo, A., 2014. Morphofunctional characterization and antibacterial activity of

698

haemocytes from Octopus vulgaris. J. Nat. Hist. 1-19, DOI:

699

10.1080/00222933.2013.826830.

700 701

Vaz-Pires, P., Seixas, P., Barbosa, A., 2004. Aquaculture potential of the common

702

octopus (Octopus vulgaris Cuvier, 1797): a review. Aquac. 238, 221-238.

703 704

Villanueva, R., Koueta, N., Riba, J., Boucaud-Camou, E., 2002. Growth and proteolytic

705

activity of Octopus vulgaris paralarvae with different food rations during first feeding,

706

using Artemia nauplii and compound diets. Aquac. 205, 269–286.

30

Figure 1. Histological sections of control specimens at the point of injection with sterile 2% NaCl. All samples, except in figure 1A, stained for immunohistochemistry and counterstained with hematoxylin. A: Normal structure of muscle tissue; B: General view on Day 0; C: View on Day 2; D: View on Day 4.

Figure 2. Histological sections of control specimens at the point of injection with sterile 2% NaCl. All samples stained for immunohistochemistry and counterstained with hematoxylin. A: View on Day 6; B: View on Day 8; C: View on Day 10; D: View on Day 12. Arrows indicate hemocytes. Arrowheads pinpoint hemocytes with pseudopodia.

Figure 3. Histological sections of infected specimens at the point of injection with the pathogen. All samples stained for immunohistochemistry (light to dark brown colour) and counterstained with hematoxylin. A: View on Day 2. Arrows indicate large hemocytes with phagocytosed antigens. **: Area with intense staining of antigens. Arrowheads indicate isolation of the area with antigen concentration; B: View on Day 2. Arrowheads indicate elongated forms of cells; C: View on Day 4. General view of the infected area. Arrowheads demarcate borderlines of reactions; D: View on Day 4. Detail of antigen presence indicated by arrows; E: High magnification view on Day 6. Arrows indicate elongated forms of cells.

Figure 4. Histological sections of infected specimens at the point of injection with the pathogen. All samples stained for immunohistochemistry (light to dark brown colour) and counterstained with hematoxylin. A: View on Day 8. ***: Area with large necrotic cells. Arrowheads indicate hemocytes; B: View on Day 12. Arrows indicate pockets with phagocytosed antigen in muscle. Arrowhead pinpoints diffuse reactions; C: Detailed view of pockets with phagocytosed antigen on Day 12; D: View on Day 14. Arrowheads indicate elongated cell forms.

Figure 5. Histological sections of gills, digestive gland and kidney of control and infected specimens. All samples stained for immunohistochemistry (light to dark brown colour) and counterstained with hematoxylin. A: Gills of control specimen; B: Gills of infected specimen; C: Digestive gland of control specimen; D: Digestive gland of infected specimen; E: Kidney of control specimen; F: Kidney of infected specimen. Arrowheads indicate hemocytes. Arrows indicate diffuse presence of antigen.

Graphical Abstract

HIGHLIGHTS · · · · ·

Intramuscular infection of common octopus with high doses of Photobacterium damsela subsp. piscicida did not caused disease. Ph. damsela subsp. piscicida provoked a rapid and very intense inflammatory response. Hemocytes phagocytosed foreign antigens and restricted the infection. There was no evidence of antigens in the gills and kidneys. The digestive gland may play a role in clearance of infectious substances.