Field studies of seahorse population density,structure and habitat use in a semi-closed north-eastern Mediterranean marine area (Stratoni,North Aegean Sea)

The present study was carried out in the marine area of Stratoni, Greece, where two seahorse species are present (Hippocampus hippocampus and Hippocampus guttulatus). Two surveys were conducted (September 2016 and May 2019) to gather information regarding seahorse species' abundance, distribution and habitat characteristics. Four different seahorse natural and artificial habitat types were identified. The results revealed that the presence of H. hippocampus was relatively high, especially at sites with artificial structures, whereas the presence of H. guttulatus was rare. Data collected can provide baseline information for future population assessments.     

Seahorse (Hippocampinae) population fluctuations in the Ria Formosa Lagoon,south Portugal

Comparisons of three sets of surveys in the Ria Formosa Lagoon, Portugal, over a 13 year period (2001–2002, 2008–2009 and 2010–2013) revealed significant population fluctuations in at least one of the two seahorse (Hippocampinae) species living there, and that those fluctuations were potentially associated with habitat changes in the lagoon. After a significant decline between the first two survey periods (2001–2002 v. 2008–2009), long‐snouted seahorse Hippocampus guttulatus populations increased significantly between 2008–2009 surveys and new 2010–2013 surveys. There were no significant differences in H. guttulatus populations between the 2001–2002 and 2010–2013 surveys. In contrast, there were no significant differences in short‐snouted seahorse Hippocampus hippocampus densities among the 16 sites surveyed throughout the three sampling periods, although the ability to detect any change was hampered by the low densities of this species in all time periods. Fluctuations in H. guttulatus densities were positively correlated with the percentage of holdfast coverage, but with none of the other environmental variables tested. These results highlight the importance of holdfast availability in maintaining stable seahorse populations. While population fluctuations are certainly more promising than a consistent downward decline, such extreme fluctuations observed for seahorses in the Ria Formosa Lagoon could still leave these two species vulnerable to any additional stressors, particularly during low density periods.     

Identifying key environmental variables of two seahorse species (<Emphasis Type="Italic">Hippocampus guttulatus</Emphasis> and <Emphasis Type="Italic">Hippocampus hippocampus</Emphasis>) in the Ria Formosa lagoon,South Portugal

Recent findings reported a significant decrease in abundance of two seahorse species (Hippocampus guttulatus and H. hippocampus) in the Ria Formosa lagoon (South Portugal) and no direct causes have been, so far, clearly identified. This study aimed to describe fluctuations in the local seahorse populations through monthly surveys over a course of a year, in order to identify some of the potential drivers behind the seasonal fluctuations. A total of six sites were chosen based on their habitat characteristics. The highest H. guttulatus abundances were recorded at sites with higher holdfast availability and depth ranging from 3 to 6 m, while H. hippocampus were observed at highest numbers in sites with lower holdfast availability and patchy distribution. In most sites, seahorse density decreased during the summer months (from May to August) and increased from August to December. Holdfast use changed across the surveyed sites, according to the respective habitat characteristics. This study identified environmental variables that influenced the abundance of seahorse population, i.e., holdfast availability, depth and temperature in the Ria Formosa lagoon, underlining the importance of monitoring populations over a course of no less than a year in order to avoid bias due to seasonal fluctuations. Identifying critical habitats will provide valuable information for local authorities in order to implement protective measures towards seahorse conservation.     

Species identification and genetic structure of threatened seahorses in Gran Canaria Island (Spain) using mitochondrial and microsatellite markers

A non-invasive DNA analysis of seahorse populations was carried out after extensive underwater surveys in Gran Canaria Island (Spain). In this geographical area, the presence of two species, Hippocampus hippocampus and H. guttulatus, has been previously reported. Sequencing of 16S ribosomal DNA (16S rDNA) was used for specific identification of live seahorses sampled in situ, as a previous step to evaluate genetic structure based on ten microsatellite markers. Phylogenetic analyses revealed the presence of a single species, H. hippocampus, in the seahorse communities found at Gran Canaria. No evidences of H. guttulatus or interspecific hybrids were found based on 16S rDNA and microsatellite data. The nuclear markers revealed low genetic diversity and lack of population structure across populations of Gran Canaria Island, with evidence of small population sizes. This study provides critical information to support conservation strategies of Gran Canaria seahorses.     

Serial monogamy in the European long-snouted seahorse Hippocampus guttulatus

Seahorses (Hippocampus spp.) are non-sex-role-reversed members of the Syngnathidae family that provide extensive brood care. Previous studies of seahorses have revealed monogamy within a single brood, but their longer term mating system had not been comprehensively evaluated. The parental contribution to 29 wild-born broods of Hippocampus guttulatus, sampled from six Portuguese populations with differing seahorse densities and sex ratios, was assessed using microsatellite DNA markers. To assess the longer term genetic mating system of this species parentage was determined in eleven broods sampled from a captive population over two breeding seasons. Genetic data suggest that this socially polygamous seahorse is serially monogamous across breeding seasons, i.e. monogamous within a season but may switch mates between seasons, and that differing population densities and sex ratios do not affect the mating system.     

Population dynamics of the long-snouted seahorse (Hippocampus guttulatus Cuvier, 1829) in the Mar Menor coastal lagoon

A 10-year monitoring program was developed to quantify the population dynamics of the long-snouted seahorse population in the Mar Menor coastal lagoon. Based on 985 underwater visual censuses, we estimated the long-snouted seahorse (Hippocampus guttulatus Cuvier, 1829) population size in the Mar Menor lagoon and its reduction in size in the last decades, as well as the effect of eutrophication crises in 2016 and 2019 on the species. The annual recruitment for the 2013–2020 period was estimated by comparing the relative abundance of early seahorse life stages in the ichthyoplankton. The density ranged from 0.0458 specimens/m³ at the beginning of the sampling period to 0.0004 at the end, showing a statistically significant difference between the three analyzed periods (H_gl=2 = 14.0, p = 0.001). The long-snouted seahorse population from the Mar Menor lagoon exemplifies the impact of fishing activities and human pressure, especially euxinic episodes and habitat destruction. As a result of this, the Mar Menor population has decreased from several million specimens to a few thousand, in only three decades. This species showed considerable resilience, the seahorse population began to recover once fishing activity stopped. In contrast, the long-snouted seahorse showed high vulnerability to habitat loss and an episodic flooding event. Adult seahorses showed preferences for highly complex habitats, especially Caulerpa prolifera–Cymodocea nodosa mixed meadows and habitats of high complexity and anthropogenic origin, such as harbors, jetties, or breakwaters. In contrast, juvenile seahorses preferred monotonous seabeds with low complexity, such as the sandy beds that are characteristic of the Mar Menor lagoon littoral.     

The use of a non‐invasive tool for capture–recapture studies on a seahorse Hippocampus guttulatus population

In this study, the spot pattern in Hippocampus guttulatus was analysed using a computer programme algorithm that allowed individual comparison. This methodology was first tested in a controlled environment using 51 adult and 55 juvenile H. guttulatus. Positive matches were obtained in 86·3 and 83·6% of the adults and juveniles, respectively. In a second experiment, monthly surveys were carried out in five selected locations in the Ria Formosa Lagoon, south Portugal, over the course of a year and a total of 980 photographs were analysed. Photographed H. guttulatus were re‐sighted one to nine times during the course of the survey period with an overall re‐sight record of over 30%. Photo‐identification was therefore shown to be a useful tool for non‐invasive mark–recapture studies that can be successfully used to survey the population abundance of H. guttulatus aged 6 months or older in consecutive years. This could be of great value when considering the assessment of H. guttulatus populations and understanding changes over time.     

An assessment of West African seahorses in fisheries catch and trade

This study provides the first assessment of a heavily traded West African seahorse species, Hippocampus algiricus, and the first information on short‐snouted seahorse Hippocampus hippocampus biology in Africa. A total of 219 seahorses were sampled from fisher catch in Senegal and The Gambia, with estimated height at reproductive activity for H. algiricus (161 mm) larger than mean ± s.d . catch height (150 ± 31 mm). Catch composition, height at reproductive activity and potential biases in fishery retention are discussed with regard to the current Convention on International Trade of Endangered Species (CITES) guidelines.     

Novel life‐history data for threatened seahorses provide insight into fishery effects

Life‐history variables for three incidentally captured species of seahorse (Kellogg's seahorse Hippocampus kelloggi, the hedgehog seahorse Hippocampus spinosissimus and the three‐spot seahorse Hippocampus trimaculatus) were established using specimens obtained from 33 fisheries landing sites in Peninsular Malaysia. When samples were pooled by species across the peninsula, sex ratios were not significantly different from unity, and height and mass relationships were significant for all species. For two of these species, height at physical maturity (H_M) was smaller than the height at which reproductive activity (H_R) commenced: H. spinosissimus (H_M = 99·6 mm, H_R = 123·2 mm) and H. trimaculatus (H_M = 90·5 mm, H_R = 121·8 mm). For H. kelloggi, H_M could not be estimated as all individuals were physically mature, while H_R = 167·4 mm. It appears that all three Hippocampus spp. were, on average, caught before reproducing; height at 50% capture (H_C) was ≥H_M but ≤H_R. The results from this study probe the effectiveness of assessment techniques for data‐poor fisheries that rely heavily on estimates of length at maturity, especially if maturity is poorly defined. Findings also question the sustainability of H. trimaculatus catches in the south‐west region of Peninsular Malaysia, where landed specimens had a notably smaller mean height (86·2 mm) and markedly skewed sex ratio (6% males) compared with samples from the south‐east and north‐west of the peninsula.     

Histological development of the long‐snouted seahorse Hippocampus guttulatus during ontogeny

The objective of the present study was to describe histological development of the European long‐snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the male's pouch. Digestive tract specialization occurred at 89 effective day‐degrees (D°_eff), corresponding to 15 days post partum (dpp), with development of the first intestinal loop and mucosal folding. At 118 D°_eff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage. The fovea emerged at 177 D°_eff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 D°_eff; 0–60 dpp), but the first oogonia were present at 30 D°_eff (5 dpp). In 354 D°_eff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 D°_eff. The present study demonstrates that approximately 89 and 177 D°_eff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1°C and an age of 1 month (177 D°_eff), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming mature at the age of 2 months (354 D°_eff).     

Specific authentication of Hippocampus based on SNP markers

Seahorses, which are used as traditional Chinese medicine, have large market demands and premium price. Because of overfishing, many seahorse species are endangered and listed in IUCN Red List. The purpose of this study was to develop a simple, rapid and reliable molecular method for specific authentication of seahorse species. Cytochrome c oxidase subunit I (COI) sequences were analyzed for molecular authentication of seahorses in this study. Many single nucleotide polymorphism (SNP) sites were detected among COI sequences of nine seahorse species. Based on the SNP sites, four pairs of modified specific primers were designed respectively for authentication of Hippocampus kuda, Hippocampus japonicus, Hippocampus trimaculatus and Hippocampus kelloggi using allele-specific PCR. Moreover, a multiplex allele-specific PCR for simultaneous authentication of H. kuda, H. japonicus and H. trimaculatus was also developed. The study showed that allele-specific PCR required no sequencing and electrophoresis, and the only necessity is to add fluorochrome into PCR products. Hence the whole authentication procedure only takes about 1.5 h. This work provides an effective method for rapid and reliable identification of seahorse species.     

Does the female seahorse still prefer her mating partner after a period of separation?

For species showing sexual monogamy, once one male and one female form a mating pair bond, they will be faithful to each other in subsequent breeding events. However, if their pair bond is broken for some reason, do they continue to prefer their partner when they come together again for mating? In other words, can the broken pair bond of sexually monogamous species be repaired? This is an interesting question but not yet well answered. To address this question, in the present study we used the lined seahorse (Hippocampus erectus), a typical sexually monogamous species, to study the partner preference of a female individual who experienced a complete separation followed by a reunion with her partner. Our main findings are as follows: (i) The female seahorse no longer prefers her partner after a separation, whether it is a former partner or a recent partner. No preference for partner-males may indicate that the broken pair bond cannot be repaired. (ii) The female seahorse maintains sexual fidelity to her partner in the absence of separation. However, once the health of her partner decreases, the female will switch mate, and her courtship with the new partner can take place during the pregnancy of her original partner. The first finding may provide insight into whether monogamous species still have an opportunity to reselect a new partner in the future to correct their poor choice once they have mated with a low-quality partner. The answer is that they can still gain an opportunity as long as the pair bonds with their current partners are broken. The second finding may reveal the conditions and timing at which a female seahorse switches her mate. These findings help us better understand the mating system of the seahorse H. erectus.     

Non-lethal dorsal fin sampling for stable isotope analysis in seahorses

Sampling collection for stable isotope analysis has traditionally involved the sacrifice of the animal. Seahorses (Hippocampus spp.) are listed as threatened by the Convention on International Trade in Endangered Species (http://www.cites.org) and consequently lethal sampling is undesirable. We evaluated the adequacy of dorsal fin tissue of adult seahorses Hippocampus guttulatus for stable isotope analysis as an alternative to lethal tissue sampling. Three seahorse tissues (dorsal fin, muscle, and liver) were analyzed for comparisons of δ¹⁵N and δ¹³C values. Similarities found between δ¹⁵N and δ¹³C values in dorsal fin and muscle tissue of H. guttulatus suggest that both tissues are adequate for stable isotope analysis to understand feeding ecology of seahorses. However, considering the threatened status of the species, dorsal fin tissue would be recommended in adult seahorses as a non-lethal sampling. The effect of lipid extraction on carbon and nitrogen stable isotope values was also evaluated in each seahorse tissues. Significant effects of lipids extraction did only occur for δ¹³C values in muscle and liver. It was found that lipid removal was not necessary to perform SIA in dorsal fin tissues. Due to the limited availability of fin tissue obtained from fin-clipping in seahorses, the relationship between the mass/surface of dorsal fin clip and stable isotope values was analyzed. δ¹⁵N and δ¹³C values in fin samples were found to be independent of the size of fin analyzed. According to our study, the use of fin-clipping sampling, with a minimum surface analyzed of 12.74?mm², was found to be an adequate method for SIA in seahorses.     

Does underwater flash photography affect the behaviour,movement and site persistence of seahorses?

The effect of flash photography on seahorse species has never been tested. An experiment was established to test the effect of flash photography and the handling of Hippocampus whitei, a medium‐sized seahorse species endemic to Australia, on their behavioural responses, movements and site persistence. A total of 24 H. whitei were utilized in the experiment with eight in each of the three treatments (flash photography, handling and control). The effect of underwater flash photography on H. whitei movements was not significant; however, the effect of handling H. whitei to take a photograph had a significant effect on their short‐term behavioural responses to the photographer. Kaplan–Meier log‐rank test revealed that there was no significant difference in site persistence of H. whitei from each of the three treatments and that flash photography had no long‐term effects on their site persistence. It is concluded that the use of flash photography by divers is a safe and viable technique with H. whitei, particularly if photographs can be used for individual identification purposes.     

Population characteristics of two seahorses, Hippocampus coronatus and Hippocampus mohnikei, around seagrass beds in the southern coastal waters of Korea

Population characteristics of two seahorse species, Hippocampus coronatus and Hippocampus mohnikei, in seagrass beds in the southern coastal waters of Korea were determined based on field observations. In Zostera beds in the Yeosu area, there was a mean density of 2.9 seahorses per 1,000?m² for H. coronatus and 1.4 seahorses per 1,000?m² for H. mohnikei. The greatest numbers of young individuals of both species were observed in July, with numbers decreasing through November. The male:female sex ratio was 1:1.6 for H. mohnikei and 1:1.7 for H. coronatus. Breeding males of H. coronatus and H. mohnikei were observed from July to November and from July to September, respectively. Within the study site, the populations of the two seahorse species exhibited low density and patchy distributions, with variation in temporal abundance being significantly correlated with water temperature. The findings of this study provide the basis for future research on the population status of H. coronatus and H. mohnikei in seagrass beds.     

Microsatellite Analysis of Olive Fly Populations in the Mediterranean Indicates a Westward Expansion of the Species

Bactrocera oleae is the major insect pest of the olive fruit. Twelve microsatellite loci isolated from the genome of this insect were used in a Mediterranean-wide population analysis. These loci were highly polymorphic with a mean number of alleles per locus of 10.42 and a mean effective number of alleles of 2.76. The analysis was performed on a sample of 671 flies collected from nineteen locations around the European part of the Mediterranean basin. Despite the high level of gene flow across the Mediterranean, results support the notion of a differentiation of three subpopulations: one of the Iberian Peninsula, one of Greece and Italy and one of Cyprus. In addition, the gradual decrease of heterozygosity from the Eastern to the Western part of the Mediterranean indicates a westward expansion of the species.     

Is filament clipping an effective tool for tissue sampling in <Emphasis Type="Italic">Hippocampus guttulatus</Emphasis>?

Examining genetic diversity, population structure, and geographic distribution has become an important part in the conservation strategies of endangered populations. However, these studies rely on tissue samples collection for DNA analysis which may be problematic for species with a sensitive conservation status. Partial fin-clipping has been employed for tissue collection, due to the increased popularity of DNA-based analysis for ecological and fisheries studies, however, fin clipping can potentially affect behavior, swimming performance, predator avoidance and the ability to find and capture prey. This study aimed to test the effects of filament clipping using captive breed seahorses (Hippocampus guttulatus) as an alternative tool for tissue sample. Clipped filaments regrew on average 0.05?±?0.02 mm/day for 3 months and no mortality or disease was observed during the experiment. Filaments provided enough tissue for DNA analysis. This study provided valuable information regarding a new sampling technique that does not impair the seahorse locomotion. This methodology may be used in population’s genetic studies of other species that have skin filaments as a morphological trait.     

Isolation and characterization of SNP variation at 90 anonymous loci in the banded wren (<Emphasis Type="Italic">Thryothorus pleurostictus)</Emphasis>

Single nucleotide polymorphisms (SNPs) are becoming more commonly used as molecular markers in conservation studies. However, relatively few studies have employed SNPs for species with little or no existing sequence data, partly due to the practical challenge of locating appropriate SNP loci in these species. Here we describe an application of SNP discovery via shotgun cloning that requires no pre-existing sequence data and is readily applied to all taxa. Using this method, we isolated, cloned and screened for SNP variation at 90 anonymous sequence loci (51 kb total) from the banded wren (Thryothorus pleurostictus), a Central American species with minimal pre-existing sequence data and a documented paucity of microsatellite allelic variation. We identified 168 SNPs (a mean of one SNP/305 bp, with SNPs unevenly distributed across loci). Further characterization of variation at 41 of these SNP loci among 256 individuals including 37 parent–offspring families suggests that they provide substantial information for defining the genetic mating system of this species, and that SNPs may be generally useful for this purpose when other markers are problematic.     

Two new species of seahorses (Syngnathidae, Hippocampus) from the Middle Miocene (Sarmatian) Coprolitic Horizon in Tunjice Hills, Slovenia: The oldest fossil record of seahorses

Two new and the only known extinct seahorse species Hippocampus sarmaticus and Hippocampus slovenicus are described from the Middle Miocene beds (Lower Sarmatian) in Slovenia, representing the oldest known fossil record of seahorses. Hippocampus sarmaticus was most similar to the extant seahorse species Hippocampus trimaculatus, while H. slovenicus can be most easily compared to the extant pygmy seahorses H. bargibanti, H. denise, and H. colemani. These Sarmatian seahorses lived among seagrasses and macroalgae in the temperate shallow costal waters of the western part of the Central Paratethys Sea.     

Does a No-Take Marine Protected Area Benefit Seahorses?

Seahorses are iconic charismatic species that are often used to ‘champion’ marine conservation causes around the world. As they are threatened in many countries by over-exploitation and habitat loss, marine protected areas (MPAs) could help with their protection and recovery. MPAs may conserve seahorses through protecting essential habitats and removing fishing pressures. Populations of White''s seahorse, Hippocampus whitei, a species endemic to New South Wales, Australia, were monitored monthly from 2006 to 2009 using diver surveys at two sites within a no-take marine protected areas established in 1983, and at two control sites outside the no-take MPA sites. Predators of H. whitei were also identified and monitored. Hippocampus whitei were more abundant at the control sites. Seahorse predators (3 species of fish and 2 species of octopus) were more abundant within the no-take MPA sites. Seahorse and predator abundances were negatively correlated. Substantial variability in the seahorse population at one of the control sites reinforced the importance of long-term monitoring and use of multiple control sites to assess the outcomes of MPAs for seahorses. MPAs should be used cautiously to conserve seahorse populations as there is the risk of a negative impact through increased predator abundance.