Histological Analysis of African and South American Lungfish Olfactory Organs

All procedures were approved by the local Animal Ethics Committee of Iwate University. The African lungfish P. aethiopicus and South American lungfish, L. paradoxa, were purchased from commercial suppliers. The fishes were anesthetized with tricaine methanesulfonate and euthanized by decapitation. Information pertaining to the animals is shown in Table 1. Juvenile and adult individuals of each lungfish were used. According to Mlewa and Green (2004) [29 (link)] and Jorgensen and Joss (2010) [30 ], P. aethiopicus individuals over 43 cm in body length (BL) reach sexual maturity. Thus, P. aethiopicus #1 (BL 50 cm) and L. paradoxa #1 (BL 65 cm) were regarded as adults, whereas P. aethiopicus #2–4 and L. paradoxa #3 (BL 35 cm or less) were regarded as juveniles [29 (link), 30 ]. Also, we confirmed during dissection whether they had functional genital organs or not.Table 1

Animals

	Animal No	Total body length (cm)	Body weight (g)	Sex	Application
P. aethiopicus	1	50.0	349.0	F	ISH (left)/RNA extraction (right)
	2	35.0	150.6	M	Dice CT
	3	31.5	100.0	unknown	ISH
	4	34.0	118.3	F	SEM
L. paradoxa	1	65.0	994.5	F	RNA extraction (left)/ISH (right)
	3	18.5	18.6	M	ISH

ISH in situ hybridization; Dice CT Diffusible iodine-based contrast-enhanced computed tomography; SEM Scanning Electron Microscopy

For histological examination, olfactory organs were dissected from the heads and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The specimens were cryoprotected in a sucrose gradient (10%, 20%, and 30% in 0.1 M PB), embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan), and sectioned sagittally using a cryostat. Sections (20 µm in thickness) were thaw mounted on MAS-coated slides (Matsunami, Osaka, Japan), air-dried, and processed for hematoxylin–eosin staining, immunohistochemistry, and in situ hybridization.