GENETIC MATING SYSTEM OF SPOTTED SEAHORSE (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.
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Abstract: 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
Introduction: 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 complicated courtship behavior; social 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 Filicampus tigris (Gronell, 1984), C. haematopterus (Matsumoto and Yanagisawa, 2001). Male seahorses cannot mate polygynously as his brood is sealed after a single eggs transfer from female because saltwater saltw 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. monogamous
Methodology: 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 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 extrac using CTAB protocol (Bruford et.al 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 pre at 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 Mera Seagrass 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 Hardy equilibrium, and genotypic disequilibrium among pairs of loci (Raymond & Rousset, 1995); Multi-locus 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).
Results: 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 expected heterozygosity and exclusion probabilities.
Genotype frequencies of both sets of males were out of the Hardy-Weinberg equilibrium (P0.05), which means there is no association between each loci and could be used as independent markers. 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.
Discussion: 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 fuscus, Vincent 1994b; Hippocampus angustus, Jones et.al., 1998; Hippocampus comes, Parente et.al.,2002; Hippocampus subelongatus, Kvarnemo et.al., 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
