Swim Bladder Tumors in The Wavy Medaka (Oryzias Latipes)

Abstract: Swim bladder tumors were detected in three out of 28 wavy medakas aged about 2 years old, all of which displayed abnormal swimming patterns caused by their spinal curvature. The tumors were located in the dorsal abdominal cavity. The swim bladder lumen was not detected in the region where it was originally assumed to be located, and that region was replaced with adipose tissue. The tumors were non-invasive, expansile, and encapsulated solid masses composed of a homogenous population of well-differentiated, densely packed, gas glandular epithelium-like cells. The tumor masses were connected to the rete mirabile, but the tumor cells did not infiltrate into them. Histopathologically, these tumors were diagnosed as adenomas originating from the gas glandular epithelium of the swim bladder. Spontaneous swim bladder tumors are rare in medaka, with an incidence of 0.02%; however, in the present study of wavy medaka, the incidence was much higher (10.7%). The long-term physical effects on the gas gland caused by swim bladder deformation considered to be a secondary effect of the spinal curvature may be an important factor in the proliferation of the gas glandular epithelium in the wavy medaka, resulting in the higher incidence of swim bladder tumors. (DOI: 10.1293/tox.2020-0058; J Toxicol Pathol 2021; 34: 107–111) 

Keywords: adenoma, gas gland, spontaneous, swim bladder, wavy medaka 

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The swim bladder in teleosts originates from an outgrowth of the anterior part of the alimentary canal and does not exist in mammals. It is the primary organ for controlling whole-body density, buoyancy, and sound production1. Anatomically, the swim bladder wall comprises two layers: the tunica interna composed of a simple flat epithelium, and the tunica exteria composed of connective tissue2, 3. The action of both a rete mirabile and a gas gland controls the volume of gas in the swim bladder. The rete mirabile is a dense bundle of parallel arterial and venous capillaries arranged side by side and utilizes countercurrent blood flow within the net to act as a countercurrent exchanger. The gas gland comprises a folded cuboidal or columnar epithelium that secretes gas into the swim bladder. In toxicity and field studies, limited pathological lesions of the swim bladder have been reported4, because this organ is not routinely examined, although it is sometimes included by chance during sagittal or transverse whole-body sectioning of smaller fish5. Additionally, since the swim bladder is often punctured and deflated during pathological preparation, its lesions are commonly overlooked.

Spontaneous swim bladder tumors are rare in teleosts, with only a few cases described in a handful of species6. Swim bladder tumors can be roughly classified into two types based on their origin: mesenchymal and epithelial tumors. The former originate from the smooth muscle and fibroblastic tissues of the swim bladder wall and are diagnosed as leiomyosarcomas7 and fibrosarcomas8, 9. These mesenchymal tumors occur in salmon and are frequently associated with a retroviral infection10. The latter originate from the swim bladder epithelium and are diagnosed as adenomas, papillary adenomas, and/or adenocarcinomas. Most spontaneous and chemical-induced swim bladder tumors in the teleosts are categorized as gas gland epithelial tumors. The epithelial tumors have been reported in medaka11, mullet12, guppy11, 13, cod14, seahorse15, and Nothobranchius fish16. In the present study, we encountered swim bladder tumors in three wavy medakas and described their detailed histopathological features.

Twenty-eight wavy medakas, aged about 2 years old, were sourced from the small stocks of wavy medaka at the Biological Research Laboratory, Nissan Chemical Corporation. These stocks were obtained via home breeding of some wavy medakas encountered naturally in the stocks maintained at the laboratory. The fish were maintained in dechlorinated tap water at 25 ± 1°C under a 16:8-hour light: dark photoperiod. The wavy medakas and one normal medaka were sacrificed by overexposure to CO2 gas and fixed in Bouin's solution overnight before being refixed in 10% neutral-buffered formalin. The fixed medakas were separated into two sections by mid-sagittal cut, and both sections were embedded in paraffin, sectioned at a thickness of 4 µm, and stained routinely with hematoxylin and eosin for histopathological examination. This study was conducted according to the Guidelines for Animal Experimentation, Biological Research Laboratory, Nissan Chemical Corporation.

Histopathology of the Swim Bladder in the Normal and Wavy Medakas 

The swim bladder in the normal medaka was located posterior and inferior to the head and body kidney, respectively, in the dorsal abdominal cavity that was divided by the diaphragm superior to the gastrointestinal tract. The shape of the swim bladders in the sagittal section was a lateral prolate spheroid shape (Fig. 1a). The wavy medakas exhibited a spinal curvature characterized by dorsoventrally curved vertebrae, resulting in abnormal swimming patterns. The swim bladders in the wavy medakas were located in the dorsal abdominal cavity, the same as in the normal medaka; however, they had a longitudinal oval shape in the sagittal section (Fig. 1b). The gas gland and rete mirabile were located in similar positions in both the normal and wavy medakas at the cranial pole of the swim bladder. The gas gland was composed of three to four layers of the pale eosinophilic vacuolated cuboidal epithelium (Fig. 1c and d) and was connected to the rete mirabile that had parallelly arranged blood capillaries (Fig. 1d).

Histopathology of Swim Bladder Tumors in the Wavy Medakas

The tumor of Fish No. 1 (male) was located posterior to the head kidney in the dorsal abdominal cavity (Fig. 2a). The other tumors of Fish No. 2 and 3 (female) were located inferior to the body kidney in the dorsal abdominal cavity and were connected to the rete mirabile (Fig. 3a and 4a). The rete mirabile of Fish No. 2 was slightly congested (Fig. 3b). In all three fish, the swim bladder lumen was not detected in the region where it was originally assumed to be located, and that region was replaced with adipose tissues (Fig. 2a, 3a and 4a). The tumor masses in these tissues were non-invasive, expansile, and encapsulated solid masses of proliferating tumor cells (Fig. 2b, 3b, and 4b). No infiltration of tumor cells into the rete mirabile had occurred (Fig. 3c and 4b). The tumor masses were composed of a homogenous population of well-differentiated, densely packed, gas glandular epithelium-like cells. The tumor cells were arranged in cords, trabeculae, and solid patterns, supported by capillaries and minimal stroma. They exhibited various sizes and were of round to polygonal shape, with distinct cell borders and pale eosinophilic vacuolated cytoplasm (Fig. 2c). Multinucleate cells and cytomegalic cells were also scattered throughout. The nuclei exhibited anti-mononucleosis, with irregularly shaped and unclear nucleoli, although no mitotic figures were detected within the tumor masses. A few foci of adipocytes were scattered throughout the tumor (Fig. 4c). Based on these features, these tumors were diagnosed as adenomas originating from the gas glandular epithelium of the swim bladder. Additionally, these tumor cells did not seem to function as gas glandular epithelium, since the swim bladder lumen did not form in these wavy medakas. With regards to histopathological lesions in other organs, there were large blood cysts in the kidney and multiple hepatic cysts with necrosis and inflammation in Fish No.1, calcification in the kidney and multiple hepatic cysts in Fish No. 2, and no lesions in Fish No. 3.

Swim bladder tumors can be induced in teleosts via exposure to environmental contaminants and carcinogens17, 18. The chemical-induced swim bladder tumors are reported in the medaka exposed to 4-chloroaniline19, aniline19, Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG)20, or bis (trin-butyl tin)oxide21; in the guppy exposed to methyl mercury chloride22; and in the rainbow trout exposed to diethylnitrosamine17, methylazoxymethanol acetate17, Benz (a)pyrene17, MNNG17, N-methyl nitrosourea, dimethylbenz[a]anthracene17, or 2,6-dimethylnltrosomorpholine23. Conversely, spontaneous swim bladder tumors are rare in teleosts, with an incidence of 0.02% (2/10,000) in medakas than 24 weeks of age and 0.14% (7/5,000) in guppies older than 13 weeks of age, which have been used in the control groups of a variety of carcinogenesis tests11. In contrast, juvenile fish with skeletal deformations show a high prevalence of spontaneous formations. In the present cases, the incidence of spontaneous swim bladder tumors was much higher at 10.7% (3/28) in the wavy medaka, compared with the normal variant. The wavy medaka develops due to a vertebral abnormality that is determined by an autosomal recessive gene (wavy; wy)25 and is characterized by wavy dorso-ventral curves in the vertebral column26, 27. There have been no reports of a relationship between the wavy gene and swim bladder tumors. In the present study, the swim bladder deformation was observed in wavy medakas, and this change was considered to be a secondary effect of the spinal curvature. Thus, the long-term physical effects of swim bladder deformation on the gas gland may be an important factor in the proliferation of the gas glandular epithelium in the wavy medaka, resulting in a higher incidence of swim bladder tumors. Furthermore, histological investigations of the gas glandular epithelium need to be conducted during the deformation process of the swim bladder in the wavy medaka.

Disclosure of Potential Conflicts of Interest: The authors declare that there is no conflict of interest. 

Acknowledgments: 

The authors would like to thank Dr. Jeffrey C. Wolf in Experimental Pathology Laboratories Inc. for some important suggestions, as well as Ms. Kaori Maejima, Ms. Hiromi Asako, Mr. Atsushi Funakoshi, Ms. Yukiko Sudo, Mr. Makoto Tsuchiya, and Mr. Yoshinori Tanaka in Nissan Chemical Co. for their excellent technical assistance.

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5. Lumsden JS. The gastrointestinal tract, swimbladder, pancreas, and peritoneum. In: Systemic Pathology of Fish: A Text and Atlas of Normal Tissue Responses in Teleosts, and Their Responses in Disease, 2nd. Ferguson HW (ed). Scotian Press, London. 187–192. 2006. 

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