Olfactory discrimination of female reproductive status by male tilapia (Oreochromis mossambicus)
1 Centro de Ciências do Mar, Universidade do Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal
2 Departamento de Biologia, Universidade de Évora, Apartado 94,
7002-554 Évora, Portugal
* Author for correspondence (e-mail: phubbard{at}ualg.pt)
Accepted 9 March 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: chemical communication, behaviour, reproduction, courtship, fish, cichlid, pheromone
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Given the range and complexity of reproductive strategies in cichlids
(Barlow, 1991;
Keenleyside, 1991
), it is
surprising that the phenomenon of chemical communication has not been equally
well investigated in this group. Ample evidence suggests that it is important
in kin recognition (Barnett,
1982
; Russock,
1990
; Vives, 1988
)
and male-male aggression (Giaquinto and
Volpato, 1997
), but the chemicals involved, and their routes of
release, are unknown. Other studies have suggested that sex steroids may also
have pheromonal roles (Oliveira et al.,
1996
; Robinson et al.,
1998
; Rocha and Reis-Henriques,
1996
,
1998
) during courtship, but
this has not been tested directly. The Mozambique tilapia (Oreochromis
mossambicus Peters 1852) can spawn repeatedly throughout the year.
Reproductively active females have a regular ovulatory cycle of 15-20 days
(Coward and Bromage, 2000
),
which is normally only interrupted by successful spawning; they are maternal
mouth-brooders, and mouth-brooding females delay their next ovulatory cycle
until the fry are released. Males establish a hierarchy where dominant
individuals develop a characteristic black colouration and aggressively defend
a nest and associated area. We have previously shown that female tilapia have
high olfactory sensitivity to substances released by males, particularly in
the urine (Frade et al.,
2002
), and that males increase their rate of urination as part of
their courtship display (Almeida et al.,
2005
). Furthermore, urine from territorial males is a more potent
odorant than that from non-territorial males
(Frade et al., 2003
). It seems
likely, therefore, that, in contrast to the goldfish, territorial male tilapia
are sending chemical signals to the females. However, in a given population,
the number of males displaying territoriality varies; a few remain territorial
on a permanent basis, a similar number remain subordinate whilst a third group
display intermittent territoriality
(Oliveira and Almada, 1996
).
Preliminary observations suggest that these males become territorial when one
or more of the females becomes pre-ovulatory; i.e. ready to spawn. This
observation suggests that males are able to assess the reproductive status of
females and alter their behaviour accordingly
(Almeida et al., 2005
). It is
possible that pre-ovulatory females release substances into the water, which
the males then detect by olfaction, and it is this chemical information that
stimulates courtship behaviour and territoriality in the males. This is the
hypothesis tested by the current study.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Recording of the electro-olfactogram (EOG)
EOGs were recorded from male tilapia exactly as previously described for
females (Frade et al., 2002).
Briefly, males were anaesthetized by immersion in water containing 100 mg
l-1 MS-222 and immobilized by intramuscular injection of gallamine
triethiodide (3 mg kg-1 in 0.9% saline). The fish were then wrapped
in a damp paper towel, gently clamped in a purpose-built V-clamp, and aerated
water containing 50 mg l-1 MS-222 was pumped over the gills
via a silicon tube placed in the mouth. The ring of cartilage
surrounding the single nostril on the right side was cut away. The olfactory
rosette was then continually irrigated with dechlorinated, charcoal-filtered
tapwater via a gravity-fed system (6 ml min-1) that
terminated in a small glass tube that was held close to the olfactory rosette.
The DC voltage was recorded by glass micropipette electrodes filled with 0.9%
NaCl in 4% agar, the recording electrode being placed close to the olfactory
epithelium near to the raphe between two adjacent lamellae. The reference
electrode was placed lightly on the skin near the nostril and connected to
earth via the headstage of the amplifier. Odorant-containing water
was introduced into this flow via a three-way valve for a period of
10 s. The resultant EOG was recorded on a personal computer running
appropriate software (Axoscope 1.1; Axon Instruments, Inc., Foster City, CA,
USA). The peak amplitude of the EOG was measured, blank-subtracted and
normalized (using the response to the `standard' 10-5 mol
l-1 L-serine) as previously described
(Frade et al., 2002
).
Normalization reduces the variation due to differences in the absolute
amplitude of the EOG recorded. This variation may be due to differences in
electrode position or conductivity of the water as well as differences in the
olfactory sensitivity of individual fish. However, the overall pattern of
responses was very similar among different fish. Blanks and standards were run
at regular intervals throughout the recording period.
Producing anosmia in males
Males were anaesthetized, immobilized and placed in the V-clamp as
described above. The rings of cartilage surrounding both nostrils were
removed, and the olfactory epithelium was cauterized with a high-temperature
fine-tip cauterizer (model AA11; Aaron Medical Industries, Inc., St
Petersburg, FL, USA). A local antiseptic was applied to the wound
(Betadine®; ASTA Medica Produtos Farmacêuticos, Lda, Lisbon,
Portugal). Sham-operated males were treated in exactly the same way, except
with no burning of the epithelium. The fish were allowed to recover for 24 h
in isolation. Behavioural experiments began the following day (48 h after
the operation). Operated fish survived well but anosmic fish had difficulty in
locating food; they would take pellets dropped into the tank whilst falling
but, once on the bottom, the pellets were ignored. Two to three weeks after
the experiment, the anosmic fish were killed and the olfactory cavity examined
under a binocular microscope; the olfactory lamellae were beginning to
regenerate.
Behaviour of anosmic and sham-operated males
An anosmic or sham-operated male was injected intramuscularly with 2.4 mg
isosulfan blue (Sigma-Aldrich Química, S.A., Sintra, Spain) in 20 µl
0.9% saline (Appelt and Sorensen,
1999) and put back in isolation. The following morning, the
behaviour of the male was video-taped for a control period of 45 min, during
which the frequency and duration of urine pulses (made visible by the
isosulfan blue) were recorded. A pre- or post-ovulatory female was then
introduced into the tank and the recording procedure repeated for a further 45
min. The fish were then returned to their family tanks. After a period of at
least 24 h, the entire experiment was repeated with a post- or pre-ovulatory
female (as appropriate) in place of the previous female. Each male was exposed
to a female from a different family tank. Male behaviour was classified as one
of the following categories (Baerends and
Baerends van Roon, 1950
;
Oliveira, 1995
) and
quantified, in terms of duration, using The Observer Video-Pro 4.0 software
(Noldus Information Technology, Wageningen, The Netherlands):
immobile - the fish remains immobile either resting on the substrate or within the water column;
swimming - active movement within the water column using the caudal fin for propulsion (but not including the behaviours outlined below);
|
|
other - coprophagia and nipping (the fish nips at the surface of the water).
Statistical analysis
Length, mass and GSI of pre- and post-ovulatory females were compared using
Student's t-test. Normalized EOG amplitudes in response to water
extracts from pre- and post-ovulatory females were compared by Student's
t-test for paired samples. Thresholds of detection were estimated by
linear regression of normalized (faeces and bile) or normalized and
log-transformed (urine and plasma) EOG amplitudes and calculation of the
intercept with the x-axis
(Hubbard et al., 2003). Linear
regressions (slopes and line elevations) were compared between body-fluid
stimuli from pre- and post-ovulatory females by Student's t-test
(Zar, 1996
). The frequency and
mean duration of urine pulses released by anosmic or sham males in the
presence of a pre- or post-ovulated female were compared by repeated-measures
ANOVA followed by the Tukey HSD test. The duration of each behavioural
category was compared by ANOVA, setting male type as the independent factor
and female type as the repeated factor. In all cases, a P value of
less than 0.05 was taken to represent statistical significance. All data are
shown as means ± S.E.M.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Olfactory responses to female body-fluids
All female body fluids evoked typical fish-form EOGs in male conspecifics,
the amplitudes of which were strongly concentration dependent
(Fig. 3). Both urine and faeces
from pre-ovulatory females evoked EOGs of significantly larger amplitude than
those from post-ovulatory females (Fig.
3A,B), whereas the responses to bile fluid and plasma from pre-
and post-ovulatory females were essentially identical
(Fig. 3C,D). Estimated
thresholds of detection were 1:105.6 and 1:105.1 for
urine, 1:106.4 and 1:106.1 for faeces (pre- and
post-ovulatory, respectively), 1:106.4 for bile and
1:105.3 for plasma.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the males gave robust olfactory responses to individual female
body fluids, clear quantitative differences were seen in the amplitude and
threshold of detection of the responses to pooled urine and faeces from pre-
and post-ovulatory females (Fig.
3A,B). This suggests that pre-ovulatory females are releasing
compounds via both urine and faeces that post-ovulatory females are
not. However, it is also possible that other important compounds are released
across the gills. As the responses to dilutions of the plasma from the two
groups of females were virtually identical
(Fig. 3C), it is unlikely that
these compounds are circulating factors, unless they are tightly bound to
circulating binding proteins and are therefore unavailable for olfactory
detection. Equally, the similarity of the responses to dilutions of the bile
fluid from the two groups of females suggests that bile acids, although potent
odorants, are not responsible for the difference in olfactory potency of the
faeces. Nevertheless, it is possible that bile acids are modified
differentially by pre- and post-ovulatory females during their transit down
the gut, with the result that pre-ovulatory females release more potent
metabolites of bile acids via the faeces. Alternatively, a recent
study has shown that social status has profound effects on the rate of release
of bile fluid into the gut (Earley et al.,
2004); subordinate male convict cichlids (Archocentrus
nigrofasciatum) retain more bile than dominant males, and this is largely
independent of food intake. This may well also alter the odour of the faeces.
Whether females of different reproductive status also retain bile fluid to
differing degrees remains to be tested.
Although the olfactory responses of females to male urine and bile fluid
(Frade et al., 2002) are
quantitatively similar to those of males to female urine and bile fluid
(present study), the thresholds of detection for faeces are over an order of
magnitude different (1:104.6, male faeces; 1:106.4 and
1:106.1, pre- and post-ovulatory female faeces, respectively). At
present, the significance of this observation is unclear, although it suggests
that females are releasing something via the faeces that the males
are not.
Behavioural responses of anosmic and sham-operated males
In the presence of pre-ovulatory females, males dramatically increase their
rate of urination (Almeida et al.,
2003) as well as displaying typical courtship behaviours such as
nest-building, quivering and circling the female
(Baerends and Baerends van Roon,
1950
). We have previously shown that male tilapia behave
differently towards pre- and post-ovulatory females
(Almeida et al., 2005
). The
major behavioural difference found was that males urinate much more frequently
in the presence of pre-ovulatory than post-ovulatory females. The
sham-operated males in the current study also dramatically increased their
rate of urination in the presence of pre-ovulatory, but not post-ovulatory,
females (Fig. 4A). Preliminary
observations suggested that the smell of females alone is not sufficient to
evoke these courtship behaviours in males (data not shown). However, anosmic
males failed to increase their rate of urination in the presence of either
pre- or post-ovulatory females (Fig.
4B). Intriguingly, other behavioural categories, including
courtship, were apparently unaffected by anosmia. This may be due, in part, to
the experimental design; males often court newly introduced, unfamiliar
females whether pre-ovulatory or not. Males in established family tanks,
however, only seem to display courtship behaviour when one of the females is
close to ovulation (A.M., O.G.A., P.C.H. and E.N.B., unpublished
observations). Furthermore, visual and olfactory input may be processed
independently by the central nervous system, at least in the context of
courtship. During the spawning season, male goldfish are able to discriminate
between male and female conspecifics using visual stimuli alone
(Thompson et al., 2004
). They
do not, however, display any courtship behaviour towards the females unless
they are in the same tank (i.e. able to smell them too). Taken together, these
results suggest that whilst the male tilapia needs to see the female in order
to display the full range of courtship behaviours, he will only invest in
chemical signalling if he can smell her and judge her ready for spawning.
Conclusions
The current study strongly suggests that pre-ovulatory female tilapia are
releasing specific odorants into the water, probably via both urine
and faeces, and that males base their behavioural response to these females,
with respect to urination frequency, on this olfactory information. The
identity of these compounds is currently under investigation.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Almeida, O. G., Barata, E. N., Hubbard, P. C. and Canário, A. V. M. (2003). Urination rate of male tilapia (Oreochromis mossambicus) is highly dependent on social context. Comp. Biochem. Physiol. A 134 (Suppl.),S27 -S28.
Almeida, O. G., Miranda, A., Frade, P., Hubbard, P. C., Barata,
E. N. and Canário, A. V. M. (2005). Urine as a social
signal in the Mozambique tilapia (Oreochromis mossambicus).
Chem. Senses 30 (Suppl.
1), i309-i310.
Appelt, C. W. and Sorensen, P. W. (1999). Freshwater fish release urinary pheromones in a pulsatile manner. In Advances in Chemical Signals in Vertebrates (ed. R. E. Johnston, D. Müller-Schwarze and P. W. Sorensen), pp.247 -256. New York: Kluwer Academic/Plenum Publishers.
Baerends, G. P. and Baerends van Roon, J. M. (1950). An introduction to the study of the ethology of cichlid fishes. Behaviour 1(Suppl.), 1-243.
Barlow, G. W. (1991). Mating systems among cichlid fishes. In Cichlid Fishes: Behaviour, Ecology and Evolution (ed. M. H. A. Keenleyside), pp.173 -190. London: Chapman and Hall.
Barnett, C. (1982). The chemosensory responses of young cichlid fish to parents and predators. Anim. Behav. 30,35 -42.
Coward, K. and Bromage, N. R. (2000). Reproductive physiology of female tilapia broodstock. Rev. Fish Biol. Fisheries 10,1 -25.
Dulka, J. G., Stacey, N. E., Sorensen, P. W. and Van Der Kraak, G. J. (1987). A steroid sex pheromone synchronizes male-female spawning readiness in goldfish. Nature 325,251 -253.[CrossRef]
Earley, R. L., Blumer, L. S. and Grober, M. S. (2004). The gall of subordination: changes in gall bladder function associated with social stress. Proc. R. Soc. Lond. B 271,7 -13.[CrossRef]
Frade, P., Hubbard, P. C., Barata, E. N. and Canário, A. V. M. (2002). Olfactory sensitivity of the Mozambique tilapia to conspecific odours. J. Fish Biol. 61,1239 -1254.[CrossRef]
Frade, P., Barata, E. N., Hubbard, P. C. and Canário, A. V. M. (2003). Olfactory detection of a putative reproductive pheromone in tilapia (Oreochromis mossambicus). Comp. Biochem. Physiol. A 134 (Suppl.),S28 .
Giaquinto, P. C. and Volpato, G. L. (1997). Chemical communication, aggression, and conspecific recognition in the fish Nile tilapia. Physiol. Behav. 62,1333 -1338.[CrossRef][Medline]
Hara, T. J. (1994). Olfaction and gustation in fish: an overview. Acta Physiol. Scand. 152,207 -217.[Medline]
Hubbard, P. C., Barata, E. N. and Canário, A. V. M.
(2003). Olfactory sensitivity to catecholamines and their
metabolites in the goldfish. Chem. Senses
28,207
-218.
Keenleyside, M. H. A. (1991). Parental care. In Cichlid Fishes: Behaviour, Ecology and Evolution (ed. M. H. A. Keenleyside), pp. 191-208. London: Chapman and Hall.
Miranda, A., Barata, E. N., Hubbard, P. C. and Canário, A. V. M. (2003). Olfactory discrimination of female reproductive status by male tilapia (Oreochromis mossambicus). Comp. Biochem. Physiol. A 134 (Suppl.),S28 .
Oliveira, R. F. (1995). Etologia social e endocrinologia comportamental da tilápia Oreochromis mossambicus (Teleostei, Cichlidae). PhD thesis. Instituto Superior de Psicologia Aplicada, University of Lisbon: Lisbon.
Oliveira, R. F. and Almada, V. C. (1996). Dominance hierarchies and social structure in captive groups of the Mozambique tilapia Oreochromis mossambicus (Teleostei, Cichlidae). Ethol. Ecol. Evol. 8,39 -55.
Oliveira, R. F., Almada, V. C. and Canário, A. V. M. (1996). Social modulation of sex steroid concentrations in the urine of male cichlid fish Oreochromis mossambicus. Hormones Behav. 30,2 -12.[CrossRef][Medline]
Pinillos, M. L., Guijarro, A. I., Delgado, M. J., Hubbard, P. C., Canário, A. V. M. and Scott, A. P. (2003). Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.). Fish Physiol. Biochem. 26,197 -210.
Robinson, R. R., Fernald, R. D. and Stacey, N. E. (1998). The olfactory system of a cichlid fish responds to steroidal compounds. J. Fish Biol. 53,226 -229.[CrossRef]
Rocha, M. J. and Reis-Henriques, M. A. (1996). Plasma and urine levels of C18, C19 and C21 steroids in an asynchronous fish, the tilapia Oreochromis mossambicus (Teleostei, Cichlidae). Comp. Biochem. Physiol. C 115,257 -264.[Medline]
Rocha, M. J. and Reis-Henriques, M. A. (1998). Steroid metabolism by ovarian follicles of the tilapia Oreochromis mossambicus (Telostei, Cichlidae). Comp. Biochem. Physiol. B 121,85 -90.[CrossRef]
Russock, H. I. (1990). The effect of natural chemical stimuli on the preferential behaviour of Oreochromis mossambicus (Pisces: Cichlidae) fry to maternal models. Behaviour 115,315 -326.
Sorensen, P. W., Hara, T. J. and Stacey, N. E.
(1987). Extreme olfactory sensitivity of mature and
gonadally-regressed goldfish to a potent steroidal pheromone,
17,20ß-dihydroxy-4-pregnen-3-one. J. Comp. Physiol.
A 160,305
-313.
Sorensen, P. W., Hara, T. J., Stacey, N. E. and Goetz, F. W.
M. (1988). F prostaglandins function as potent olfactory
stimulants that comprise the postovulatory female sex pheromone in goldfish.
Biol. Reprod. 39,1039
-1050.
Stacey, N. and Sorensen, P. (2002). Hormonal pheromones in fish. In Non-Mammalian Hormone-Behavior Systems, vol. 2 (ed. D. W. Pfaff, A. P. Arnold, A. M. Etgen, S. E. Fahrbach and R. T. Rubin), pp.375 -434. London: Harcourt Publishers Ltd/Academic Press.
Thompson, R. R., George, K., Dempsey, J. and Walton, J. C. (2004). Visual sex discrimination in goldfish: seasonal, sexual, and androgenic influences. Hormones Behav. 46,646 -654.[CrossRef][Medline]
Vermeirssen, E. L. M. and Scott, A. P. (1996). Excretion of free and conjugated steroids in rainbow trout (Onchorhynchus mykiss): Evidence for branchial excretion of the maturation-inducing steroid, 17,20ß-dihydroxy-4-pregnen-3-one. Gen. Comp. Endocrinol. 101,180 -194.[CrossRef][Medline]
Vermeirssen, E. L. M. and Scott, A. P. (2001). Male priming pheromone is present in bile, as well as in urine, of female rainbow trout. J. Fish Biol. 58,1039 -1045.[CrossRef]
Vives, S. P. (1988). Parent choice by larval convict cichlids, Cichlasoma nigrofasciatum (Cichlidae, Pisces). Anim. Behav. 36,11 -19.
Zar, J. H. (1996). Biostatistical Analysis. New Jersey: Pearson Higher Education.
Zhang, C., Brown, S. B. and Hara, T. J. (2001). Biochemical and physiological evidence that bile acids produced and released by lake char (Salvelinus namaycush) function as chemical signals. J. Comp. Physiol. B 171,161 -171.[Medline]
Related articles in JEB: