Sense of Smell

Data were obtained from 9139 subjects [4928 females aged 5–96 years (M = 31.8, SD = 18.9) and 4211 males aged 5–91 years (M = 30.7, SD = 17.7)]. Among them, 3432 (37.5%) had been included in a previous study to establish normative data [15 (link)]. According to the inclusion criteria for the respective studies, all subjects were healthy and none reported histories for any olfactory disturbances.
Odors were delivered using felt-tip pens (“Sniffin’ Sticks”) of approximately 14 cm length and an inner diameter of 1.3 cm. These pens carry a tampon soaked with 4 ml of liquid odorant. For odor presentation, the cap was removed from the pen for approximately 3 s, the pen’s tip brought in front of the subject’s nose and carefully moved from left to right nostril and backwards [3 (link)].
The threshold was obtained in a three alternative forced choice paradigm (3 AFC) where subjects were repeatedly presented with triplets of pens and had to discriminate one pen containing an odorous solution from two blanks filled with the solvent. Phenylethanol (dissolved in propylene glycol) or n-butanol (dissolved in water) were used, with both odorants having been found equivalent in olfactory sensitivity testing: scores obtained with both are correlated [17 (link)]. The highest concentration was a 4% odor solution. Sixteen concentrations were created by stepwise diluting previous ones by 1:2. Starting with the lowest odor concentration, a staircase paradigm was used where two subsequent correct identifications of the odorous pen or one incorrect answer marked a so-called turning point, and resulted in a decrease or increase, respectively, of concentration in the next triplet. Triplets were presented at 20 s intervals. The threshold score was the mean of the last four turning points in the staircase, with the final score ranging between 1 and 16 points.
The discrimination task used the same 3 AFC logic. Two pens of any triplet contained the same odorant, while the third pen smelled differently. Subjects were asked to indicate the single pen with a different smell. Within-triplet intervals were approximately 3 s. As the odors used in this subtest were more intense, between-triplets intervals were 20–30 s. The score was the sum of correctly identified odors. Hence, the scores in this task ranged from 0 to 16 points. Importantly, subjects were blindfolded for the threshold and discrimination tasks to avoid visual identification of target pens.
Odor identification comprised common and familiar odorants (recognized by at least 75% of the population). Subjects were presented with single pens and asked to identify and label the smell, using four alternative descriptors for each pen. Between-pen intervals were approximately 20–30 s. The total score was the sum of correctly identified pens, thus subjects could score between 0 and 16 points.
The final “TDI score” was the sum of scores for Threshold, Discrimination and Identification subtests, with a range between 1 and 48 points.

The nucleotide sequences of primers specific to the V1R genes of P. aethiopicus and L. paradoxa are shown in Table 2. cDNA was synthesized from total RNA derived from the olfactory organs using oligo dT primers and ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. PCR was performed using the cDNA as the template together with Ex Taq (Takara, Shiga, Japan). The PCR products were analyzed by 1.5% agarose gel electrophoresis.Table 2

Primers for V1Rs

Probe name	Target V1R gene	Product size (bp)	Forward primer (5 > 3)	Reverse primer (5 > 3)
Paeth01	P. aethiopicus V1R23 (ancV1R)	597	CCCACAGTTAGCTGGCGTAA	GGTTTTGGCATGCCTCATGG
Paeth02	P. aethiopicus V1R52	403	CATTGGTTTGACCTGCCTGC	CTCTGCTCCAGCTTCCTGAC
Paeth03	P. aethiopicus V1R53	566	AGCCTAGCATGCTCAAACCT	ACCACCATCTTGGATGCCTG
Paeth04	P. aethiopicusV1R55	551	TGCTGTTGGCCTTGCAAGTA	TTGCCACAGCCATAAGGACT
Paeth05	P. aethiopicusV1R69	599	TGCTAAGCTGCTTCCAGTGT	AGAGTGGCAAGTCACTGCAT
Paeth06	P. aethiopicusV1R71	658	CTTCTGACTGGGGGTGTTCC	CCAAGGACAGAAAATGCCGC
Paeth07	P. aethiopicusV1R83	596	ACTTGCCAACCCACCAAGAA	GAAATGCAACGTCACGAGCA
Paeth08	P. aethiopicusV1R94	563	CGTGTTTGTCGAGCGATGTC	GCAAAGAAGACACGGGCATC
Paeth09	P. aethiopicusV1R103	545	CTTTTCACGCTGGGACTTCC	GTGACAACAGTCTTGGCAGC
Paeth10	P. aethiopicusV1R111	543	GGGGCAAACCTGTTACTCCT	TGCTTGTAGCTCTGCTGTGG
Paeth11	P. aethiopicusV1R116	523	CGAGAGGCATTCCTGAACCA	TTAGCTGCCTGACCTTCTGC
Paeth12	P. aethiopicusV1R119	402	GACAAGTACTGGTGTTCTGGGT	TAAAGGAGCAGGCCACAACA
Paeth13	P. aethiopicusV1R136	477	TGATCCTTTGCAACCTGGGA	ACAAAATGTTGCTGCTGGCC
Paeth14	P. aethiopicusV1R140	589	CCGTGTTTTTCGAGCGATGT	AGGACTGACAGCAGCATACA
Paeth15	P. aethiopicusV1R141	513	CCAGAGGAATGCCACAGACA	CCCTGGCTTCAGCTGAAACA
Paeth16	P. aethiopicusV1R144	432	CACTGAACTGGCAGGGACAA	ATCAGGTCACGGGCAAAACT
Paeth17	P. aethiopicusV1R148	645	TCAGAGCTGTCAGTGGCAAA	CCGTGACACTGATGCCTGAT
Paeth18	P. aethiopicusV1R159	393	CAAGTACTGGAGTCTTGGGCA	GCAGGCCACAATGCATAACC
Paeth19	P. aethiopicusV1R160	695	TGGAAACATCACATCCGGCA	TGCTTGCTTCTCTGCTGTGA
Paeth20	P. aethiopicusV1R166	525	TACCCGAGGTCTTCCAGCAA	GCTGCTTTCACCTCTACAGC
Paeth21	P. aethiopicusV1R198	410	GTAGTAAGCGGCATCCCTGG	ACAGTGTACATTGGTGGGCT
Paeth22	P. aethiopicusV1R208	527	GGTTGTGCTGACAGTAGGCA	CTTGGGCTTCTGCACTGTTC
Paeth23	P. aethiopicusV1R213	549	ATGGTTGCTTTGCTGTCACG	TACAACCGACTTTGCAGCCT
Paeth24	P. aethiopicusV1R218	539	AGCTTCACAAAAGGGGCCAT	GCAAAGCCGTTCACCTGAAA
Paeth25	P. aethiopicusV1R227	520	CAAGAGGGGTTCCAGACTGT	GCTGCTCTGTTCTCTGCTGT
Paeth26	P. aethiopicusV1R257	305	CTCTGTGTGCTTGCTATGGC	ACTGTTTTTGCTGCTTGGCC
LP01	L. paradoxa V1R20 (ancV1R)	599	TACTGTTAGCTGGCGCAACA	TCTGCGTTTGGGGATTCCTC
LP02	L. paradoxa V1R59/L. paradoxa V1R60	620	AATGAGCTGCCCCAAACTGA	AGGTGACAACAGTTCGCGTA
LP03	L. paradoxa V1R64	640	ACAGTTACTGGAGCTGTGGG	TCTCTGCACTGTTCTCCAGC
LP04	L. paradoxa V1R65	543	ACCTGTCAACAGCAAACCTGA	TTGCTGCTTGACTCTCTGCA
LP05	L. paradoxa V1R70	525	TGCAAGAGGAGTGCCACAAA	GATTTTGCTGCCCTGGCTTC
LP06	L. paradoxa V1R80	552	ACCAGCAAACCTCACCATCA	ATGTAGCTGCTGGCAAGTGT
LP07	L. paradoxa V1R89	554	TTGCTGTCCGGAGTAAACCT	GCTGCTTGGCTTTCTGCATT
LP08	L. paradoxa V1R92/L. paradoxa V1R93	490	GGATCAGTGTCCTGGACAGC	TGAGGTCACGGCCAAAAAGA
LP09	L. paradoxa V1R99	652	CAGGTCTCTCTGGGGACTGA	AGGCAAAGTGTTGAGGCAGT
LP10	L. paradoxa V1R103/L. paradoxa V1R104	510	ACTTGGCCATCACTGGATCC	CCACCATGAGATCTCGGCTG
LP11	L. paradoxa V1R120/L. paradoxa V1R121	439	TCACATCCCACCTTGCTTTT	ATTACAGCATCACGCCCTGT
LP12	L. paradoxa V1R127	707	CTGCCCATGGTCTTCTCCAA	AATGGGGTCTCACCTGTTGC
LP13	L. paradoxa V1R130	556	TCCTGCCAACATTGCCATCT	AAAAAGGATTGCTGCGCTGG
LP14	L. paradoxa V1R139	412	TATCACGCGGCATGGCTATT	GACTCGGTGCGATCCTTCAT
LP15	L. paradoxa V1R142	524	AGTGTGTGAGTGTCAGTGCA	TGCAGCATAGCACATCGAGA
LP16	L. paradoxa V1R172	540	GAGCTGCTTCCAGTATGCCA	ACTGAAGCATAGCACGTGGA

For RNA probe synthesis, each PCR product was subcloned into the pCRII-TOPO vector using the TOPO TA Cloning Kit Dual promotor (Thermo Fisher Scientific). Next, the sequence of each clone was verified. Closely related V1R genes cannot be distinguished due to their high sequence identity, so some probes (LP02, LP08, LP10, and LP11) were expected to detect multiple V1R genes (Table 2).

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.