(Hippocampus kuda, Bleeker, 1852)
Ooi Boon Leong, Juanita Joseph, Choo Chee Kuang
Department of Marine Science, Faculty of Maritime Studies & Marine Science, Universiti Malaysia
Terengganu, 21030 Kuala Terengganu, Terengganu.
[email protected], [email protected], [email protected]
Seahorse, from the family Syngnathidae, is unique in sexual reproduction where males get pregnant by
carrying and incubating the eggs rather than the female. Many studies on seahorse reproductive behaviors
were carried out ex-situ and on temperate species which showed that these seahorses mate monogamously.
This research focused on Hippocampus kuda at the Merambong seagrass bed off the Pulai River estuary in
Johor. We observed different embryonic development stages in some clutches from the preliminary
sampling, thus suggesting the possibility of polygamous mating in the species. Samplings were carried out
between May-November 2008 and February and March 2009. Seahorses were captured by hand using
visual censes at low spring and neap tides. The brood samples (30-35 embryos per pregnant male) were
siphoned out using capillary tube. Seahorses were tagged using Visual Implant Fluorescence Elastomer
(VIFE) and released back at the very same spot they were found. Fin clips of pregnant seahorses were also
sampled as references to paternal genotype. The samples were then extracted using CTAB protocol and
amplified by PCR using microsatellite primers developed for other seahorse species (Hcau11, Hcau36,
Habd3 and Habd9). A total of 20 broods were extracted but only 15 broods were successfully amplified.
Our data showed that only one female had contributed eggs to each clutch of all the 15 broods. This results
was further confirmed using multi-locus approach and re-confirm using GERUD 2.0. Our analysis showed
that Hippocampus kuda had mated monogamously.
KEYWORDS: Spotted seahorse, Hippocampus kuda, microsatellite, monogamous, mating system
Seahorse, from the family Syngnathidae,
is a unique fish, where the male get pregnant by
carry and incubate the egg rather than the female
(Kuiter, 2003). Many studies have been carried
out in the laboratory on the reproduction
behavior of temperate seahorse species (Vincent,
1990, 1994a,b, 1995; Masonjones & Lewis,
1996, 2000; Masonjones, 2001) which show
these seahorses mated monogamously. Few
studies however were carried out in tropics (E.g.
Hippocampus comes: Perante et al., 2002)
Reproductive success, defined as the
total offspring that are able to reach maturity and
to breed, offspring produce for mating, number
of mating events per season, adults’ reproductive
life span and the chances of offspring to survive
and reach maturity, is crucial in any kind of
organism as those who failed will cease to exist. .
Due to those conditions, many organisms have
adapted themselves to evolve. This reproductive
behavior is a key component in studies ranging
from the theoretical basis for sexual selection
and mating behavior to the models of population
dynamics (Clutton-Brock, 1988; Vincent & Giles,
2003) and can be a powerful evolutionary force
to promote traits that increase the reproductive
success through the mechanisms of mate
competition or choice (Darwin 1871; Anderson
1994; Wilson & Smith, 2007).
Conventionally, females often allocate
more energy from egg production to parental
care, sometimes they even nourish and protect
the young until reaching sexual maturity, which
was found in the mammalian worlds (CluttonBrock, 1991; Vincent & Giles, 2003). As for fish,
planktonic stage larvae is common, but if female
produce larger game, and when the parental care
existed, often these parental care will be taken by
males, thus, increase the male investment in
reproduction, rather than just as a gamete
supplier (Ridley, 1978; Baylis, 1981; Sargent &
Gross, 1985; Vincent & Giles, 2003). The
involvement of males towards the parental care
will significantly increase the survival of the
offspring (Cole & Sadovy, 1995).
In Syngnathidae family, males have the
most complex brooding structure and providing
high level of parental care by brood the eggs,
rather then females (Vincent et al., 1994b).
Extensive studies on behavioral and genetic have
been carried out on their mating system, and
studies shows that syngnathid fishes have
monogamy (one partner), social polygamy
(multiple partners), conventional
ventional sex role (males
compete for mate) and sex role reversal (female
compete for mate). Field studies shows that most
seahorse species exhibited strict monogamy
during courtship (Foster & Vincent, 2004) and
this also applies to at least two species of
pipefishes e.g. Corythoichthys intestinalis and
haematopterus (Matsumoto and Yanagisawa,
polygynously as his brood is sealed after a single
eggs transfer from female because saltwater
intrusion would spoil the eggs (Vincent, 1990b),
and males would risk receiving only a partial
clutch of eggs if he had mated with a bigamous
female (Vincent and Sadler, 1995). However, a
conflicting situation arises as Mi et al. (1998)
argued that males H. kuda can simultaneously
carry clutches from multiple females. This
observation was also noted in our previous
observation whereby different embryonic
development stages were observed in a single
clutch thus suggesting the possibility of
polygamous mating
ating in the species, which
prompted this study to speculate whether
Hippocampus kuda is indeed monogamous.
Study site
The selected study site is situated in Pulai River
River Estuary that host the largest intertidal
seagrass bed known in Peninsular Malaysia
(around 1.8 km in length and size of 38 hectares)
dominated by Enhalus acroroides
Sample collection
Sampling was carried out between MayMay
November 2008 and February and March 2009.
Seahorses were captured by hand using visual
censes at low spring and neap tides. The brood
samples (30-35 embryos per pregnant male)
were siphoned out using capillary tube and all
samples were preserved in 95% Absolute
Ethanol. Seahorses were then tagged using
Visual Implant Fluorescence Elastomer (VIFE)
and released back the very same spot they were
found. Fin clips (Approx.
Approx. 5mm2) of pregnant
seahorses were also sampled as references to
paternal genotype following Lourie et al (2005).
DNA extraction and Yield Determination
Whole embryos and adult fin clip was extracted
using CTAB protocol (Bruford 1992) with
some modification. DNA samples were eluted in
50 µl at the end of the extraction. For
microsatellite assessment, we used 4 previously
characterized seahorse loci; Habd3, Habd9
(Wilson and Smith, 2007) and Hcau11, Hcau36
(Galbusera et al., 2007). Temperature
optimization was conducted for these 4 primers
and optimum temperature was obtained.
Polymerase Chain Reaction (PCR) was
performed in Bio-Raid
Raid DNA engine machines.
10 µl reaction consisted of 1 x Promega Taq
buffer, 3.5-4.5
4.5 mM MgCl2, 0.2 µm of each
primer, 0.125 mM of each dNTP’s mix, and 0.02
units Promega Taq polymerase. The thermal
cycling proceeded by 5 min at pre-denaturing
94oC, 30 seconds of 94oC denaturing, an optimal
annealing temperature for 35
3 seconds and 72oC
of extension for 35 second. 35 cycles was
employed followed by final extension for 3
minutes at 72oC. The loci Habd3 and Hcau36
were amplified with 3.5mM MgCl2 with
annealing temperature of 48oC and 58oC,
whereas Habd9 and Hcau11 were amplified with
4.5 nM MgCl2 with annealing temperature of
54oC and 48oC respectively. Fifteen broods (269
total embryos) were genotyped successfully
amplified together with father.
Statistical analysis
Figure 1: Location map of Merambong
Bed at the Pulai River mouth
Maternity data was analyzed using three
different approaches. GENEPOP was used to
compute the exact test for Hardy-Weinberg
equilibrium, and genotypic disequilibrium
among pairs of loci (Raymond & Rousset, 1995);
locus approach (DeWoody et al., 2000)
was used to reconstruct the maternal genotypes
to identify the mother to the offspring, and rere
confirmed using GERUD (Jones, 2001).
In total, 20 broods sample have been obtained.
Out of 20 broods, only 15 broods that manage to
extract and amplified. Whereas the other 5
broods was sampled prematurely. Due to the
reason, it did not provide useable embryos for
genetic analysis. In all the 15 broods, genotypes
within the clutches were consistent with a single
mother, which the genotype of each mother were
reconstructed using multi-locus approach (table 1)
and reconfirm using GERUD (Jones, 2001).
Results shows that monogamous mating system
in seahorse, Hippocampus kuda, which is
consistent with the previous finding that males
only receive eggs from one female during
pregnancy (Jones et al., 1998)
All four cross-amplified microsatellite loci were
polymorphic, which displayed from 6- 10 alleles
per locus (Figure 1). Observed heterozygosities
was moderate, which have the value of 0.800 for
Habd3, 0.733 for Habd9, 0.600 for Hcau11 and
0.717 for Hcau36, and expected heterozygosity
high with the value of 0.819 (Habd3),
0.900(Habd9), 0.814(Hcau11) and 0.907(Hcau36)
Table 1: Summary statistic for 4 polymorphic
microsatellite loci for spotted seahorse. Shown
are locus, alleles observed (n=15), observed and
Genotype frequencies of both sets of males were
out of the Hardy-Weinberg equilibrium (P<0.05),
and there was no evidence of genotypic linkage
disequilibrium between any of the loci (P>0.05),
which means there is no association between
each loci and could be used as independent
Exclusion probability (Table1),
probability of neither parents know were 0.41 for
loci Habd3, 0.56 for loci Habd9, 0.4 for loci
Hcau11 and 0.54 for loci Hcau36. As for one
parent known with certainty, one unknown, the
probability for loci Habd3 were 0.59, loci Habd9
were 0.72 and 0.58 and 0.70 for loci Hcau11 and
loci Hcau36 respectively.
Figure 1: Allele frequency histogram for 4 microsatellite loci in Hippocampus kuda. Allelic designations
represent size in base pair (bp) of the amplified product.
Seahorse is known to have monogamous mating
system, and yellow seahorse, Hippocampus kuda
in this study show that genetic monogamy is a
common mating pattern and the results is
consistent with previous behavioral observation
of social monogamy in other seahorse species
Hippocampus angustus, Jones, 1998;
Hippocampus comes, Parente,2002;
Hippocampus subelongatus, Kvarnemo,
2000; Hippocampus abdominalis, Wilson and
Smith, 2007,). In order to meet Hardy-Weinberg
equilibrium model, 7 conditions have to be
followed, which include no mutation, no
migration, no genetic drift, no natural selection,
random mating, the population is infinitely large
and everyone produce the same amount of
offspring (Raymond & Rousset, 1995). The
result shows that it was out of the HardyWeinberg equilibrium (P<0.05), even with high
heterozygosity between each locus with the
average of 0.860 (Table 1). The probable
explanation towards this phenomenon may due
to the small population size of spotted seahorse
at Merambung Seagrass Bed.
Evolution of the brood pouch system of
monogamous mating system in seahorses.
Because of seahorse have enclosed brood pouch,
and fertilization occur internally in the brood
pouch (Ah-King, 2006); it may only
receive one clutch of eggs per brooding time.
Although difference embryonic development
was observed in previous sampling, but studies
shows that those eggs are come from the same
mother, as confirmed by microsatellite analysis.
The differential development in the embryos
may due to its different speed of development or
and also may cause by unhealthy eggs that
provided by the female that stunted the
development of the embryos.
Monogamy is a tedious way of
reproduction, and it must be sustained by natural
selection and the long-term pair bond must
overweight its cost. Monogamous mating system
seems to occur at lower densities, have reduced
mobility and fix home range (Parente,
2002). Due to that, individual of both sexes must
place much of the time to find a partners with
similar reproductive capacity or eggs or brood
pouch space will be wasted (Jones, 2003).
But studies show that when monogamy was
employed, interbrood interval was shorten after
few generation produced, means through
monogamous mating system, it will be tedious
for the initial stage, but after a few generations,
the organism can continuously reproduce
without needing extra times to find for another
mate for the next brooding period (Kvarnemo, 2000). Others seahorse study have found
that when a pair is formed, the pair’s capacity to
produce offspring was increase with time elapsed
since the pair was form (Vincent, 1994b).
Table 2: Summary of assayed males, each row represents the results for a single clutch from a single male
pregnancy. Shown in table is the male ID, paternal genotype, number of embryos genetically assayed per
clutch, and the maternal genotypes (reconstructed from the progeny array).
In conclusion, this genetic study shows
that spotted seahorse, Hippocampus kuda was
mate monogamously. This finding is consistent
with others behavioral studies on related
seahorse species that have been proven to have
social monogamy mating system.
This study was funded by by the Ministry of
Science, Technology and Innovation (MOSTI)
Fundamental Research Grant FRGS 59023. We
would like to thank the volunteers of Save Our
Seahorse (S.O.S) for helping out with the
sampling, and the Faculty of Maritime Studies
and Marine Sciences of University Malaysia
Terengganu for its logistic support.
[1] Andersson M (1994) Sexual Selection. Princeton
University Press, Princeton, New Jersey.
[2] Ah-King, M., Elofsson, H., Kvarnemo, C.,
Rosenqvist, G., Berglud, A., Where is there no
sperm competition in a pipefish with externally
brooding males? Insight from sperm activation
and morphology. Journal of Fish Biology 68,
[3] Baylis, J. R. (1981). The evolution of parental care
in fishes, with reference to Darwin’s rule of male
sexual selection. Environmental Biology of
Fishes 6, 223–251.
[4] Bruford, M.W., Hanotte, O, Brookfield, J.F.K and
Burke, T. (1992) Single-locus and multi-locus
finger printing. Pp225-269 in Hoelzel, A.L. (ed.)
Molecular genetic analysis of population – A
practical approach. Oxford University Press, NY.
[5] Clutton-Brock, T.H. (1988). Reproductive Success.
Studies of Individual Variation in Contrasting
Breeding Systems. Chicago, IL: The University
of Chicago Press
[6] Cole, K. S. & Sadovy, Y. (1995). Evaluating the
use of spawning success to estimate reproductive
success in a Caribbean reef fish. Journal of Fish
Biology 47, 181–191.
[7] Darwin, C. (1871) The Descent of Man, and
Selection in Relation to Sex. J. Murray, London.
[8] DeWoody, J.A., Walker, D., & Avise, J.C. (2000)
Genetic parentage in large half-sib clutches:
theoretical estimates and empirical appraisals.
Genetics 154:1907-1912.
[9] Fiedler, K. (1955). Vergleichende Verhaltensstudien an Seenadeln, Schlangennadeln und
Seepferdchen (Syngnathidae). Zeitschrift Fur
Tierzuchtung Und Zuchtungsbiologie11, 358–416
(in German)
[10] Foster, S.J. & Vincent, C.J. (2004) Review paper:
Life history and ecology of seahorse:
implications for conservation and management.
Journal of fish biology. 65, 1-61
[11] Galbusera, P.H.A.., Gillemot, S., Jouk, P., Teske,
P.R., Hellemans, B. & Volckaert, F.A.M.J. (2007)
Isolation of microsatellite markers for the
endangered Knysna seahorse Hippocampus
capensis and their use in the detection of a
genetic bottleneck, Molecular Ecology Notes
[12] Jones, A.G., Avise, J.C. (1997) Microsatellite
analysis of maternity and the mating system in
the Gulf pipefish Syngnathus scovelli, a species
with male pregnancy and sex-role reversal.
Molecular Ecology, 6, 203–213.
[13] Jones, A.G., Kvarnemo, C., Moore, G.I.,
Simmons, L.W. & Avise, J.C. (1998)
Microsatellite evidence for monogamy and sexbiased recombination in the western Australian
seahorse, H. angustus. Molecular Ecology. 7,
1497 – 1505
[14] Jones, A. G. (2001) GERUD 1.0: a computer
program for the reconstruction of parental
genotypes from progeny arrays using multi-locus
DNA data. Mol. Ecol. Notes 1, 33.
[15] Jones, A.G., Moore, G.I., Kvarnemo, C., Walker,
D., Avise, J.C., (2003) Sympatric speciation as a
consequence of male pregnancy in seahorse.
PNAS, vol.100, no.11
[16] Kuiter, Rudie H. (2003). Seahorses, pipefishes
and their relative, a comprehensive guide to
Syngnathiformes. pp.2. TMC publishing,
Chorleywood, UK.
[17] Kvarnemo, C., Moore, G.I., Jones, A.G., Nelson,
W.S. & Avise, J.C. (2000) Monogamous pair
bonds and mate switching in the Western
Australian seahorse, Hippocampus subelongatus.
J.Evol. Biol. 13 882-888.
[18] Lourie, S.A., M. Green and A.C.J. Vincent. 2005.
Dispersal, habitat differences, and comparative
phylogeography of Southeast Asian seahorses
Ecology. 14:1073-1094.
[19] Masonjones, H. & Lewis, S. M. (1996). Courtship
behaviour in the dwarf seahorse. Hippocampus
zosterae. Copeia 1996, 634–640
[20] Masonjones, H. & Lewis, S. M. (2000).
Differences in potential reproductive rates of
male and female seahorses relative to courtship
roles. Animal Behaviour 59, 11–20.
[21] Masonjones, H. D. (2001). The effect of social
context and reproductive status on the metabolic
rates of dwarf seahorses (Hippocampus zosterae).
Comparative Biochemistry and Physiology A 129,
[22] Mi, P.T., E.S. Kornienko and A.L. Drozdoz. 1998.
Embryonic and larval development of the
seahorse Hippocampus kuda . Russian Journal of
Marine Biology. 24(5):325-329.
[23] Perante, N.C.; Pajaro, M.G.; Meeuwig, J.J. and
Vincent, A.C.J.(2002) Biology of a seahorse
species, Hippocampus comes in the central
Philippines. Journal of Fish Biology 60, 821–837
[24] Raymond, M. & Rousset, F., (1995) GENEPOP
Version 1.2: population genetic software for
exact tests and ecumenicism. J.Hered. 86:248249
[25] Ridley, M. (1978). Paternal care. Animal
Behaviour 26, 904–932
[26] Sargent, R.C.& Gross,M.R.(1985). Evolution of
male and female parental care in fishes.
American Zoologist 25, 807–822.
[27] Vincent, A. C. J. (1990a). Reproductive ecology
of seahorses. PhD thesis, University of
Cambridge, U.K.
[28] Vincent, A.CJ. 1990b. A seahorse father makes a
good mother. Natural History. 12:34-42.
[29] Vincent, A. C. J. (1994a). Operational sex ratios
in seahorses. Behaviour 128, 153–167.
[30] Vincent, A. C. J. (1994b). Seahorses exhibit
conventional sex roles in mating competition,
despite male pregnancy. Behaviour 128, 135–151.
[31] Vincent, A. C. J. (1995). A role for daily
greetings in maintaining seahorse pair bonds.
Animal Behaviour 49, 258–260.
[32] Vincent, A.C.J. and L.M. Sadler. 1995. Faithful
pair bonds in wild seahorses, Hippocampus
whitei. Animal Behaviour. 50:1557-1569.
[33] Vincent, A.C.J. & Giles, B.G. (2003) Correlates
of reproductive success in a wild population of
Hippocampus whitei. Journal of fish biology, 63,
[34] Vincent, A.C.J., Marsden, A.D., Evans, K.L.,
Sadler, L.M. (2004) Temporal and spatial
opportunities for polygamy in a monogamous
seahorse, Hippocampus whitei. Behaviour, 141,
[35] Wilson, A.B. & Smith, K.M.M. (2007) Genetic
monogamy despite social promiscuity in the potbellied seahorse (Hippocampus abdominalis).
Molecular Ecology 16, 2345-2352