The olfactory organs of two extant lungfish, Protopterus amphibius and P. dolloi, which retain primitive characteristics and have the ability to breathe air, were investigated and compared to those of other amphibious fishes that possess excellent tolerance to being out of water. Based on morphological and histological study, the olfactory organs of the two lungfish species revealed the following primitive characteristics: i) an elongated OC with several lamellae, ii) an AN at the ventral tip of the upper lip, iii) a PN at the palate of the oral cavity, iv) a MOO with longitudinal lamellae, v) lamellae consisting of ORNs, SCs, BCs, LCs, and MCs, and vi) a VEC made up of the GE and the CSE, as reported in other lungfishes (Derivot 1984; Nakamuta et al. 2013; Wittmer and Nowack 2017). Among these features, the elongated lungfish OC resembles those of amphibious mudskippers of the subfamily Oxudercinae (Kuciel et al. 2013). In particular, the Asian swamp eel Monopterus albus possesses a pipe-like chamber penetrating the dorsal snout (Kim 2018). Nevertheless, there are some differences in the organ structure between lungfishes and other amphibious fishes. First, the two lungfishes have several folded lamellae ascending from the inner wall of the OC, whereas mudskippers have no such suspended lamellae (Kuciel et al. 2013; Kim et al. 2019). In other lungfish, the number of lamellae per one rosette row was 8 to 37 in adult P. annectens (80–550 mm in total body length) and 2 to 26 in adult Lepidosiren paradoxa (80–560 mm) (Nakamuta et al. 2013; Wittmer and Nowack 2017). Unfortunately, we could not obtain useful statistical data on the lamellae due to a shortage of specimens (only two lungfish per species). Nevertheless, we confirmed that that P. amphibius and P. dolloi have at least 20 lamellae each. In this respect, although mudskippers and lungfishes both successfully live a terrestrial life with aerial exposure, the dependance on the sensory organ might be different between two. More advanced mudskippers commonly have remarkably large eyes as an adaption to terrestrial activity (Sayer 2005; Jaafar and Murdy 2017), whereas lungfishes, which estivate in a cocoon when exposed to air, possess unremarkable “degenerate” eyes (Marshall et al. 2011).
Second, the two lungfishes possess no accessory nasal sac or any appendage for olfactory ventilation, unlike mudskippers that have one or two sacs (Kuciel et al. 2013). In amphibious fish, the accessory nasal sac generates a suction force toward the OC by increasing the internal pressure and helps take up water in shallow stagnant water or on wet land with little water (Nevitt 1991; Kuciel 2013). Unlike mudskippers, the OC of the two lungfishes has no ANS but penetrates toward the oral cavity. We hypothesize that the much larger oral-cavity volume of P. amphibius and P. dolloi, relative to ANS-containing amphibious fishes, is used for odorant residue elimination.
Of particularly interest is the distribution of MCs in the two lungfish species studied here: MCs are abundant in both the lamellae and chamber inner wall in P. amphibius but are scarce and only found in the lamella in P. dolloi. In fish skin, the mucus secreted by MCs has a large range of physiological functions: i) protection against harmful chemical or physical factors, ii) disease resistance, iii) ion and water regulation, and iv) respiratory gas exchange (Shephard, 1994). In addition, Horn and Riegle (1981) and Laming et al. (1982) found that enhanced mucous secretions prevent water loss in stichaeoid fish and shanny (L. pholis) exposed to aerial conditions. In this view, P. amphibius, with more abundant MCs than P. dolloi, can be regarded as having a different cytological strategy for aerial exposure of the olfactory organ.
Histochemically, the properties of the MCs in teleost olfactory organ can be classified into acidic or neutral mucopolysaccharide and glycoprotein according to the mixed ratio of protein and carboxylate. As the two lungfishes show a deep red color with AB (pH 2.5)-PAS but a faint color with H&E, it was concluded that MCs have only a neutral mucopolysaccharide with less ionized amino acids (Gona 1979).
The GE of the VEC in P. amphibius and P. dolloi was different from the MCs of the MOO: reddish-brown vs. faint with H&E and Masson’s trichrome and dark violet vs. dark red with AB (pH 2.5)-PAS. These features of the GE are due to acid and neutral mucopolysaccharide with much protein mass, as in other lungfish species with much granule protein (Nakamuta et al. 2013; Wittmer and Nowack 2017). Nevertheless, additional approaches are needed for identifying proteins.
Wittmer and Nowack (2017) reported two types of secretary glands, a glandular duct cell and an elongated glandular cell (glandular epithelium), but we confirmed only one type of secretory gland, the GE, in the VEC to produce mucus. The GE with AB (pH 2.5)-PAS reaction has been variably expressed in lungfishes: PAS positive but Alcian blue negative in P. annectens (Nakamuta et al. 2013), Alcian blue (pH 2.5) positive but PAS negative in L. paradoxa, and both Alcian blue and PAS positive in P. annectens (Wittmer and Nowack 2017). Tierney (2015) stated that glandular secretion of proteins is not important in fish olfaction. Meanwhile, Wittmer and Nowack (2017) suggested that protein-containing mucopolysaccharide participates in the sensory mechanism at least in the lungfish olfactory system, and the Alcian blue-positive GE functions to remove odorant residues with acidic mucus. Based on our results, we think that the VEC is deeply related to lungfish olfaction.
Consequently, we confirmed that the difference in density of MCs in P. amphibius and P. dolloi reflects different degrees of aerial exposure during their lives, and acid and neutral mucopolysaccharide-containing VEC is evolutionary evidence for lungfishes as the closest living relative to tetrapods, at least in the order Dipnoi.