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.     

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.     

Life history of an unusual marine fish: survival, growth and movement patterns of Hippocampus guttulatus Cuvier 1829

The life history of the long‐snouted seahorse Hippocampus guttulatus was characterized using mark‐recapture data collected within a focal study site and catch data from 53 additional sites in the Ria Formosa coastal lagoon, southern Portugal. Population structure in benthic habitats was characterized by high local densities (0·3–1·5 m^?2), equal sex ratios and few juveniles <70 mm. Adult H. guttulatus maintained small (19·9 ± 12·4 m²), strongly overlapping home ranges during multiple reproductive seasons. Recruited (benthic) juveniles exhibited significantly lower site fidelity than adults. A Ford‐Walford plot of standard length (L_S) at time t against L_S measured during the previous year from tagged juveniles and adults led to estimates of the von Bertalanffy parameters K = 0·571 and L_∞ = 197·6 mm. The growth rate of planktonic juveniles (inferred from previous studies), was greater than predicted by the von Bertalanffy model, providing evidence of an ontogenetic shift in growth trajectory. The instantaneous rate of natural mortality, M, ranged from 1·13 to 1·22 year^?1(annual survival rate = 29·4–32·2%). Sexes did not differ in movement, growth or survival patterns. On average, H. guttulatus measured 12·2 ± 0·8 mm at birth. Planktonic juveniles recruited to vegetated habitat at 96·0 ± 8·0 mm (0·25 years), had mature brood pouches (males only) at 109·4 mm (0·49 years), began maintaining home ranges and reproducing at 125–129 mm (0·85–0·94 years), and lived for 4·3–5·5 years. Early age at maturity, rapid growth rates, and short generation times suggested that H. guttulatus may recover rapidly when direct (e.g. exploitation) and indirect (e.g. by‐catch and habitat damage) effects of disturbance cease, but may be vulnerable to extended periods of poor recruitment.     

Optimization of hydrolysis conditions, isolation, and identification of neuroprotective peptides derived from seahorse Hippocampus trimaculatus

Hippocampus trimaculatus is one of the most heavily traded seahorse species for traditional medicine purposes in many countries. In the present study, we showed neuroprotective effects of peptide derived from H. trimaculatus against amyloid-β₄₂ (Aβ₄₂) toxicity which are central to the pathogenesis of Alzheimer’s diseases (AD). Firstly, H. trimaculatus was separately hydrolyzed by four different enzymes and tested for their protective effect on Aβ₄₂-induced neurotoxicity in differentiated PC12 cells. Pronase E hydrolysate exerted highest protection with cell viability value of 88.33 ± 3.33 %. Furthermore, we used response surface methodology to optimize pronase E hydrolysis conditions and found that temperature at 36.69 °C with the hydrolysis time 20.01 h, enzyme to substrate (E/S) ratio of 2.02 % and pH 7.34 were the most optimum conditions. Following several purification steps, H. trimaculatus-derived neuroprotective peptides (HTP-1) sequence was identified as Gly-Thr-Glu-Asp-Glu-Leu-Asp-Lys (906.4 Da). HTP-1 protected PC12 cells from Aβ₄₂-induced neuronal death with the cell viability value of 85.52 ± 2.22 % and up-regulated pro-survival gene (Bcl-2) expressions. These results suggest that HTP-1 has the potential to be used in treatment of neurodegenerative diseases, particularly AD. Identification, characterization, and synthesis of bioactive components derived from H. trimaculatus have the potential to replace or at least complement the use of seahorse as traditional medicine, which further may become an approach to minimize seahorse exploitation in traditional medicine.     

Life history and ecology of the elusive European short‐snouted seahorse Hippocampus hippocampus

To improve the understanding of the life history and ecology of one of Europe's most elusive fishes, the short‐snouted seahorse Hippocampus hippocampus, data from wild populations in a shallow coastal lagoon in southern Portugal were analysed. The data were collected from 17 tagged seahorses on a focal‐study grid as well as from >350 seahorses encountered during underwater visual surveys and a fishery‐independent study using beach seines. These populations of settled juveniles and adults had a mean population density of 0·009 m^?2. During the study period (2000–2004), reproduction peaked in July and August. Juveniles recruited to the lagoon at c. 66 mm standard length (L_S) and 0·5 years of age and established small home ranges (0·8 to 18·2 m²). First reproduction was estimated at 100 mm and 1 year of age. Based on a fitted von Bertalanffy model, H. hippocampus grew quickly (growth coefficient K = 0·93) to a maximum theoretical size L_∞ = 150 mm and have a maximum lifespan of c. 3·2 years. Courtship behaviours were consistent with the maintenance of pair bonds and males brooded multiple batches of young per year. Estimated annual reproductive output averaged 871 young (±632). Together these analyses provide the first life‐history parameters for this species and indicate that H. hippocampus bears characteristics of opportunist and intermediate strategists. Such populations are predicted to exhibit large fluctuations in abundance, making them vulnerable to extended periods of poor recruitment.     

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.     

Conservation value of Codium tenue habitat for the endangered Knysna seahorse Hippocampus capensis

We investigated the density and size structure of the endangered Knysna seahorse Hippocampus capensis in the macroalga Codium tenue at three sites in a residential marina estate in the Knysna Estuary, South Africa, over an 18 month period from March 2017 to August 2018. Seahorses were present in C. tenue throughout the year. Average (± SE) densities of 0.33 (± 0.03) and 0.23 (± 0.03) seahorses per kg of C. tenue were found for 2017 and 2018 respectively. Density did not vary across sites but varied significantly across months with a decreasing trend from summer to spring. The average (± SE) adult seahorse height was 75.16 mm (± 0.63 mm) and 69.09 mm (± 0.64 mm) and mass was 2.05 g (± 0.04 g) and 1.65 g (± 0.04 g) for males and females, respectively, and an even sex ratio was observed throughout the sampling period. Height varied across months, with significantly smaller seahorses found in June and August of 2017. Tail length was highly correlated with height and therefore can serve as a potential proxy for determining the height of H. capensis. This study showed that C. tenue is consistently utilised by Knysna seahorse and should therefore be considered an important habitat to protect and facilitate the ongoing conservation of this endangered seahorse species.     

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R_A) and standard (R_S) metabolic rate at different life stages in zebrafish Danio rerio (5 day‐old larvae, 2 month‐old juveniles and 6 month‐old adults). Scaling exponents and constants were assessed for standard (R_S = 0·273M^0·965 in mgO₂ g^?1 h^?1) and active metabolic rate (R_A = 0·799M^0·926 in mgO₂ g^?1 h^?1). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.     

Reproductive parameters of rhinobatid and urolophid batoids taken as by‐catch in the Queensland (Australia) east coast otter‐trawl fishery

Reproductive variables are provided for batoids regularly taken as by‐catch in the east coast otter‐trawl fishery on the inner‐mid continental shelf off the south‐east and central coasts of Queensland, Australia. Total length at maturity (L_T50 and 95% c.i .) for the eastern shovelnose ray Aptychotrema rostrata was 639·5 mm (617·6–663·4 mm) for females and 597·3 mm (551·4–648·6 mm) for males. Litter size (n = 9) ranged from nine to 20 (mean ± s.e. = 15·1 ± 1·2). This species exhibited a positive litter size–maternal size relationship. Disc width at maturity (W_D50 and 95% c.i .) for the common stingaree Trygonoptera testacea was 162·7 mm (155·8–168·5 mm) for females and 145·9 mm (140·2–150·2 mm) for males. Gravid T. testacea (n = 6) each carried a single egg in the one functional (left) uterus. Disc width at maturity (W_D50 and 95% c.i .) for the Kapala stingaree Urolophus kapalensis was 153·7 mm (145·1–160·4 mm) for females and 155·2 mm (149·1–159·1 mm) for males. Gravid U. kapalensis (n = 16) each carried a single egg or embryo in the one functional (left) uterus. A single female yellowback stingaree Urolophus sufflavus carried an embryo in each uterus. A global review of the litter sizes of shovelnose rays (Rhinobatidae) and stingarees (Urolophidae) is provided.     

Age,growth and maturity of the pelagic thresher Alopias pelagicus and the scalloped hammerhead Sphyrna lewini

Indonesia has the greatest reported chondrichthyan catches worldwide, with c.110,000 t caught annually. The pelagic thresher (Alopias pelagicus) and scalloped hammerhead (Sphryna lewini) together comprise about 25% of the total catches of sharks landed in Indonesia. Age and growth parameters were estimated for A. pelagicus and S. lewini from growth‐band counts of thin‐cut vertebral sections. Alopias pelagicus (n = 158) and S. lewini (n = 157) vertebrae were collected from three Indonesian fish markets over a 5 year period. A multi‐model analysis was used to estimate growth parameters for both species. The models of best fit for males and females for A. pelagicus was the three‐parameter logistic (L_∞ = 3169 mm L_T, k = 0·2) and the two‐parameter von Bertalanffy models (L_∞ = 3281 mm L_T, k = 0·12). Age at maturity was calculated to be 10·4 and 13·2 years for males and females, respectively, and these are the oldest estimated for this species. The samples of S. lewini were heavily biased towards females, and the model of best fit for males and females was the three‐parameter Gompertz (L_∞ = 2598 mm L_T, k = 0·15) and the two‐parameter Gompertz (L_∞ = 2896 mm L_T, k= 0·16). Age at maturity was calculated to be 8·9 and 13·2 years for males and females, respectively. Although numerous age and growth studies have previously been undertaken on S. lewini, few studies have been able to obtain adequate samples from all components of the population because adult females, adult males and juveniles often reside in different areas. For the first time, sex bias in this study was towards sexually mature females, which are commonly lacking in previous biological studies on S. lewini. Additionally, some of the oldest aged specimens and highest age at maturity for both species were observed in this study. Both species exhibit slow rates of growth and late age at maturity, highlighting the need for a re‐assessment of the relative resilience of these two globally threatened sharks at current high levels of fishing mortality throughout the eastern Indian Ocean.     

Some observations on the biology of two rarely seen deep‐sea chimaerids,Chimaera carophila and Hydrolagus homonycteris

Chimaera carophila (n = 45) and Hydrolagus homonycteris (n = 11), two deep‐sea chimaerids rarely caught in the waters off New Zealand, were collected from research trawl catches and commercial fishery catches around New Zealand at depths between 400 and 1300 m, between 2014 and 2016. Additional preserved specimens of both species (n = 58) from museum collections were analysed for size, sex and maturity. External assessment of male claspers and a combination of internal assessments of female gonad mass and oviducal gland width, were used to determine maturity. For both species, length at first maturity was 0·70–0·82 of their maximum observed chimaera length (L_C), with females maturing at a larger size. Length at maturity for C. carophila (L_C range: 28·7–103·9 cm) was estimated at 72·5 cm L_C for males (n = 163) and 82·5 L_C for females (n = 58). In H. homonycteris, length at maturity (length range: 78·6–99·8 cm L_C) was estimated at 79·1 cm L_C for males (n = 51) and 80·1 cm L_C for females (n = 17). Ovarian fecundity was up to 31 for C. carophila and sperm storage was confirmed in the oviducal gland of this species. Both species preyed on benthic invertebrates. Some C. carophila and H. homonycteris inhabit depths beyond most current fisheries, but both species appear to be relatively rare and have reproductive parameters characteristic of low productivity, which may make these species vulnerable to population decline if mortality was to increase in the future.     

Hypoxia tolerance decreases with body size in red drum Sciaenops ocellatus

Mass‐specific oxygen consumption rate, i.e. standard metabolic rate (R_s) and critical oxygen tension (P_crit) of red drum Sciaenops ocellatus were measured and scaled over a 2500‐fold range in mass (M_F; 0·26–686 g). R_s conformed to well established models (R_s = 3·73·91 M_F^?0·21; r² = 0·86) while P_crit increased over the size range (P_crit = 3·15 log₁₀M_F + 16·19; r² = 0·44). This relationship may be ecologically advantageous as it would allow smaller S. ocellatus to better utilize hypoxic zones as habitat and refuge from predators.     

Estimated genetic parameters for growth‐related traits in large yellow croaker Larimichthys crocea using microsatellites to assign parentage

To estimate the heritabilities of growth‐related traits in large yellow croaker, Larimichthys crocea, three independent full‐factorial crosses were created by crossing four males × four females, seven males × three females and four males × three females (set 1, set 2 and set 3). At 13 months post‐hatch, the juveniles were collected from three cross sets and measured for body mass (M), standard length (L_S) and body height (H_B). In addition, the parentage of the juveniles was assigned by genotyping six or seven polymorphic microsatellite loci. Out of the 959 juveniles, 99·6% could be assigned to a single parental pair. Heritabilities of growth‐related traits were estimated for individual and combined sets with the pedigrees reconstructed by using microsatellite genotypes. The heritability estimates at 13 month‐old were 0·02–0·30 for M, 0·02–0·25 for L_S and 0·03–0·36 for H_B. These results showed that the heritabilities of M, L_S and H_B were different among three sets, which suggested that different breeding strategies should be adopted for different sets.     

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.     

De Novo Transcriptome Analysis of Two Seahorse Species (Hippocampus erectus and H. mohnikei) and the Development of Molecular Markers for Population Genetics

Seahorse conservation has been performed utilizing various strategies for many decades, and the deeper understanding of genomic information is necessary to more efficiently protect the germplasm resources of seahorse species. However, little genetic information about seahorses currently exists in the public databases. In this study, high-throughput RNA sequencing for two seahorse species, Hippocampus erectus and H. mohnikei, was carried out, and de novo assembly generated 37,506 unigenes for H. erectus and 36,113 unigenes for H. mohnikei. Among them, 17,338 (46.23%) unigenes for H. erectus and 17,900 (49.57%) for H. mohnikei were successfully annotated based on the information available from the public databases. Through comparing the unigenes of two seahorse species, 7,802 candidate orthologous genes were identified and 5,268 genes among them could be annotated. In addition, gene ontology analysis of two species was similarly performed on biological processes, cellular components, and molecular functions. Twenty-four and twenty-one unigenes in H. erectus and H. mohnikei were annotated in the biosynthesis of unsaturated fatty acids pathways, and both seahorses lacked the Δ12 and Δ15 desaturases. Total of 8,992 and 9,116 SSR loci were obtained from H. erectus and H. mohnikei unigenes, respectively. Dozens of SSR were developed and then applied to assess the population genetic diversity, as well as cross-amplified in a related species, H. trimaculatus. The H_O and H_E values of the tested populations for H. erectus, H. mohnikei, and H. trimaculatus were medium. These resources would facilitate the conservation of the species through a better understanding of the genomics and comparative genome analysis within the Hippocampus genus.     

Standard metabolism and growth dynamics of laboratory‐reared larvae of Sardina pilchardus

This study provides the first measurements of the standard respiration rate (R_S) and growth dynamics of European sardine Sardina pilchardus larvae reared in the laboratory. At 15° C, the relationship between R_S (µl O₂ individual^?1 h^?1) and larval dry mass (M_D, µg) was equal to: R_S = 0·0057(±0·0007, ± s.e.)·M_D^{0·8835(±0·0268)}, (8–11% M_D day^?1). Interindividual differences in R_S were not related to interindividual differences in growth rate or somatic (Fulton's condition factor) or biochemical‐based condition (RNA:DNA).     

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.     

Growth and energy budget of F2'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F₂‘all‐fish’ growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29·2° C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I_E), and the proportion of I_E utilized for heat production (H_E) were significantly higher in the transgenics than in the controls. The proportion of I_E directed to waste products [faecal energy (F_E) and excretory energy loss (Z_E+U_E) where Z_E is through the gills and U_E through the kidney], and the proportion of metabolizable energy (M_E) for recovered energy (R_E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I_E= 19·3 F_E+ 6·0 (Z_E+U_E) + 45·2 H_E+ 29·5 R_E or 100 M_E= 60·5 H_E+ 39·5 R_E. The average energy budget equation of the controls was: 100 I_E= 25·2 F_E+ 7·4 (Z_E+U_E) + 35·5 H_E+ 31·9 R_E or 100 M_E= 52·7 H_E+ 47·3 R_E. These findings indicate that the high growth rate of ‘all‐fish’ transgenic common carp relative to their non‐transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.     

Genetic heterogeneity in the Indian stocks of seahorse (<Emphasis Type="Italic">Hippocampus kuda</Emphasis> and <Emphasis Type="Italic">Hippocampus trimaculatus</Emphasis>) inferred from mtDNA cytochrome b gene

Genetic stock structure analysis of two seahorse species from the south east and south west coasts of India (37 samples of Hippocampus kuda and 39 samples of Hippocampus trimaculatus from Kollam (Kerala) and Mandapam (Tamil Nadu)) was carried out through sequence variation analysis of a 350 bp cytochrome b fragment of mitochondrial DNA. This was taken up to support the breeding and restocking programme of these species in natural habitats for conservation purpose. The occurrence of strong genetic subdivision among the samples, detected by the analysis of molecular variance (AMOVA), and significant Φ_ST values indicated that stocks of both the species in the two Indian coasts are distinct. The findings of the present study have important implications for conservation and management of these two species and we recommend stock-specific, breeding assisted sea-ranching programme for H. kuda and H. trimaculatus along the Indian coasts. Handling editor: K. Martens