Olfactory responses of male and female Mozambique Olfactory responses of male and female Mozambique
Tilapia (
Tilapia (Oreochromis Oreochromis mossambicus mossambicus Peters 1952) to a Peters 1952) to a putative urinary pheromone that signals male dominance putative urinary pheromone that signals male dominance
T. Keller
T. Keller (1,2) (1,2) , A. Iacovella , A. Iacovella (1,3) (1,3) , P.C. Hubbard , P.C. Hubbard (1) (1) , A.V.M. Canario , A.V.M. Canario (1) (1) , E.N. Barata , E.N. Barata (1,2) (1,2)
(1)
CCMAR Centre of Marine Science, University of Algarve, Faro Portugal;
(2)Department of Biology, University of Évora, Portugal;
(3)Faculty of Science, University of Messina, Italy
¾Tilapia males establish dominance hierarchies; females mate preferentially with dominant territorial males
¾ Dominant males increase urination frequency during courtship and aggression (Barata et al., 2008. JCE)
¾ A sulphated amino-sterol urinary odorant for females has been suggested to act as pheromonal signal of dominance
Introduction Introduction
Do both sexes detect the same urinary odorants from males of dif
Do both sexes detect the same urinary odorants from males of different social rank? ferent social rank?
Results Results Methods Methods
Assessment of the olfactory activity of male urine, and corresponding SPE and HPLC fractions in males and females
Conclusion Conclusion
Acknowledgement
AcknowledgementFundação para a Ciência e Tecnologia (FCT, Portugal) Ph.D. fellowship SFRH/BD/46192/2008
Both males and females detect the same dominance pheromone!
Both males and females detect the same dominance pheromone!
¾ Most likely, increased release of the amino-sterol signals dominance to both sexes, but in a blend comprising other unidentified urinary odorants.
Next steps Next steps
¾ Mass (MS)- and structure (NMR) analysis of the amino-sterol like dominance pheromone
¾ Bioassay guided separation and fractionation of further (more hydrophilic) urinary odorants
Urine samples 1 volume Solid-Phase Extraction
C18 SPEcartridges
EOG recordings using tilapia males and females 1:10,000 v/v dilution Flow-through
Extract 1 volume methanol Reverse phase HPLC
C18 column water/methanol (15-100%) gradient
Peak detection Evaporative Light Scattering Detector
Fraction collection
6 Dominants 5 Intermediates 6 Subordinates
Computerised data acquisition and analysis system
Recording of the Electro-Ofactogram
Recording electrode
Olfactory rosette Reference
electrode Amplifier
Filter
Stimulus Delivery System
Anaesthetised male or female
Sample collection and processing
17 males of different dominance status (index) 17 males of different dominance status (index)
Females to SPE C18 Flow-through Males to SPE C18 Flow-through Males to SPE C18 extracts Males to untreated urine
Dominance index (arcsin-transformed values)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Normalized EOG amplitude[log(x+1)-transformed values]
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Females to untreated urine Females to SPE C18 extract
EOG responses of both sexes to urine and SPE fractions from males of different social rank
¾EOG responses of males and females were similar.
¾The olfactory potency of the three stimulus types increased with the social rank of male donor (regression lines and dashed 95% CL lines).
¾The raw urine was the most potent stimulus. The extract had a regression line with similar slope, but lower elevation (P< 0.001).
¾The flow-through was the least potent stimulus with a regression line of lower slope (P< 0.001)and elevation (P<
0.001)than raw urine and extract.
Male to Fraction 1 & 2 Male to Fraction 22 Female to Fraction 22 Female to Fraction 1 & 2
Relative peak area [log(x+1) transformed values]
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Normalized EOG amplitude[log(x+1) transformed values]
0.0 0.1 0.2 0.3 0.4 0.5
0.6 Male to SPE C18 extract Female to SPE C18 extract
EOG responses of both sexes to urine extracts and its HPLC fractions from males of different social rank
¾The urine extract and corresponding HPLC fractions 22 containing this peak (putative amino-sterol) evoked concentration-dependent EOG responses in both sexes (regression lines and dashed 95% CL lines).
¾EOG responses to fraction 22 had a similar regression line slope to that of the urine extract, but a lower elevation (P< 0.001).
¾In contrast, the EOG amplitudes elicited by another HPLC fraction (fractions 1 & 2) containing several non- retained peaks) were notcorrelated with its total peak area.
-40 160 360 560 760 960
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96 time [min]
ELSD signal [mV]
b ) Intermediate donor male
1 23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2223 24 25 26 27 28 29 30 FC
CDCA 0.4 mM -40
160 360 560 760 960
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96 time [min]
ELSD signal [mV]
a ) Dominant donor male
1 23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2223 24 25 26 27 28 29 30 FC
CDCA 0.4 mM internal standard internal standard
-40 160 360 560 760 960
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96 time [min]
ELSD signal [mV]
c ) Subordinate donor male
1 23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2223 24 25 26 27 28 29 30 FC
CDCA 0.4 mM
Example chromatograms of male urinary extracts
¾
¾HPLC of urine extracts revealed a well retained peak (RT = 65 min; fraction 22).
¾The area of this peak was larger in dominant males than in subordinates.
¾The total area of non-retained peaks in fractions 1 & 2 did notcorrelated with males’ social rank.
