Hippocampus Abdominalis Research Articles

Host-intestinal microbiota interactions in Edwardsiella piscicida-induced lethal enteritis in big-belly seahorses: novel insights into the role of Carbohydrate-Active enzymes and host transcriptional responses

Host-intestinal microbiota interactions in Edwardsiella piscicida-induced lethal enteritis in big-belly seahorses: novel insights into the role of Carbohydrate-Active enzymes and host transcriptional responses

Transcriptomic Analysis of Hippocampus abdominalis Larvae Under High Temperature Stress.

Acute temperature stress was explored in Hippocampus abdominalis through a comprehensive RNA-seq analysis. RNA-seq was conducted on 20-day-old H. abdominalis after 24 h of temperature stress. Four experimental conditions were established: a control group (18 °C) and three temperature treatment groups (21, 24, and 27 °C). Seahorse larvae were found to be unaffected by 21 °C and 24 °C and were able to survive for short periods of time during 24 h of incubation, whereas mortality approached 50% at 27 °C. The sequencing process produced 75.63 Gb of high-quality clean data, with Q20 and Q30 base percentages surpassing 98% and 96%, respectively. A total of 141, 333, and 1598 differentially expressed genes were identified in the 21, 24, and 27 °C groups vs. a control comparison group, respectively. Notably, the number of up-regulated genes was consistently higher than that of down-regulated genes across all comparisons. Gene Ontology functional annotation revealed that differentially expressed genes were predominantly associated with metabolic processes, redox reactions, and biosynthetic functions. In-depth KEGG pathway enrichment analysis demonstrated that down-regulated genes were significantly enriched in pathways related to steroid biosynthesis, terpenoid backbone biosynthesis, spliceosome function, and DNA replication. Up-regulated genes were enriched in pathways associated with the FoxO signaling pathway and mitophagy (animal). The results indicated that temperature stress induced extensive changes in gene expression in H. abdominalis, involving crucial biological processes such as growth, biosynthesis, and energy metabolism. This study provided key molecular mechanisms in the response of H. abdominalis to temperature stress, offering a strong basis for future research aimed at understanding and mitigating the effects of environmental stressors on marine species.

Anti-Photoaging Effects of Antioxidant Peptide from Seahorse (Hippocampus abdominalis) in In Vivo and In Vitro Models.

Overexposure to ultraviolet (UV) radiation can lead to photoaging, which contributes to skin damage. The objective of this study was to evaluate the effects of an antioxidant peptide (SHP2) purified from seahorse (Hippocampus abdominalis) alcalase hydrolysate on UVB-irradiated skin damage in human keratinocyte (HaCaT) and human dermal fibroblast (HDF) cells and a zebrafish model. The data revealed that SHP2 significantly enhanced cell viability by attenuating apoptosis through the reduction of intracellular reactive oxygen species (ROS) levels in UVB-stimulated HaCaT cells. Moreover, SHP2 effectively inhibited ROS, improved collagen synthesis, and suppressed the secretion of matrix metalloproteinases (MMPs) in UVB-irradiated HDF cells. SHP2 restored the protein levels of HO-1, Nrf2, and SOD, while decreasing Keap1 expression in UVB-treated HDF, indicating stimulation of the Keap1/Nrf2/HO-1 signaling pathway. Furthermore, an in vivo study conducted in zebrafish confirmed that SHP2 inhibited photoaging by reducing cell death through the suppression of ROS generation and lipid peroxidation. Particularly, 200 µg/mL of SHP2 exerted a remarkable anti-photoaging effect on both in vitro and in vivo models. These results demonstrate that SHP2 possesses antioxidant properties and regulates skin photoaging activities, suggesting that SHP2 may have the potential for use in the development of cosmetic products.

Androgens and androgen receptor directly induce the thickening, folding, and vascularization of the seahorse abdominal dermal layer into a placenta-like structure responsible for male pregnancy via multiple signaling pathways

Seahorses exhibit the unique characteristic of male pregnancy, which incubates numerous embryos in a brood pouch that plays an essential role in enhancing offspring survivability. The pot-belly seahorse (Hippocampus abdominalis) possesses the largest body size among seahorses and is a significant species in Chinese aquaculture. In this study, we revealed the cytological and morphological characteristics, as well as regulatory mechanisms, throughout the entire brood pouch development in H. abdominalis. The brood pouch originated from the abdominal dermis, extending towards the ventral midline. As the dermal layers thicken, the inner epithelium folds, the stroma loosens, and vascularization occurs, culminating in the formation of the brood pouch. Furthermore, through transcriptomic analysis of brood pouches at various developmental stages, 8 key genes (tgfb3, fgf2, wnt7a, pgf, mycn, tln2, jund, ccn4) closely related to the development of brood pouch were identified in the MAPK, Rap1, TGF-β, and Wnt signaling pathways. These genes were highly expressed in the pseudoplacenta and dermal layers at the newly formed stage as examined by in situ hybridization (ISH). The angiogenesis, densification of collagen fibers, and proliferation of fibroblasts and endothelial cells in seahorse brood pouch formation may be regulated by these genes and pathways. Additionally, the expression of the androgen receptor gene (ar) was significantly upregulated during the formation of the brood pouch, and ISH confirmed the expression of the ar gene in the dermis and pseudoplacenta of the brood pouch, highlighting its role in the developmental process. Androgen and flutamide (androgen receptor antagonist) treatments significantly accelerated the formation of the brood pouch and completely inhibited its occurrence respectively, concomitant to the upregulated expression of differentially expressed genes involved above signaling pathways. These findings demonstrated that formation of the brood pouch is determined by androgen and the androgen receptor activates the above signaling pathways in the brood pouch through the regulation of fgf2, tgfb3, pgf, and wnt7a. Interestingly, androgen even induced the formation of the brood pouch in females. We firstly elucidated the formation of the seahorse brood pouch, demonstrating that androgens and their receptors directly induce the thickening, folding, and vascularization of the abdominal dermal layer into a placenta-like structure through multiple signaling pathways. These findings provide foundational insights to further exploring the evolution of male pregnancy and adaptive convergence in viviparity across vertebrates.

Comprehensive biochemical analysis and nutritional evaluation of fatty acid and amino acid profiles in eight seahorse species (Hippocampus spp.)

Seahorses are increasingly recognized for their nutritional potential, which underscores the necessity for comprehensive biochemical analyses. This study aims to investigate the fatty acid and amino acid compositions of eight seahorse species, including both genders of Hippocampus trimaculatus, Hippocampus kelloggi, Hippocampus abdominalis, and Hippocampus erectus, to evaluate their nutritional value. We employed Gas Chromatography-Mass Spectrometry (GC-MS) and High-Performance Liquid Chromatography (HPLC) to analyze the fatty acid and amino acid profiles of the seahorse species. GC-MS was used to detect 34 fatty acid methyl esters, while HPLC provided detailed amino acid profiles. GC-MS analysis demonstrated high precision with relative standard deviations (RSDs) generally below 2.53 %, satisfactory repeatability (RSDs from 6.55 % to 8.73 %), and stability (RSDs below 2.82 %). Recovery rates for major fatty acids ranged from 98.73 % to 109.12 %. HPLC analysis showed strong separation of amino acid profiles with theoretical plate numbers exceeding 5000. Precision tests yielded RSDs below 1.23 %, with reproducibility and stability tests showing RSDs below 2.73 % and 2.86 %, respectively. Amino acid recovery rates ranged from 97.58 % to 104.66 %. Nutritional analysis revealed significant variations in fatty acid content among the species. Female H. erectus showed higher levels of hexadecanoic acid and saturated fatty acids, while male H. abdominalis had lower concentrations of n-3 full cis 4,7,10,13,16,19-docosahexaenoic acid (DHA). Total lipid yields varied from 3.2491 % to 12.3175 %, with major fatty acids constituting 17.9717 %–74.6962 % of total lipids. In conclusion, this study provides essential insights into the fatty acid and amino acid composition of seahorses, supporting their potential as valuable dietary supplements. The differences between genders in specific fatty acids suggest a nuanced nutritional profile that could be exploited for targeted dietary applications. Further research is needed to explore the seasonal and environmental variations affecting seahorse biochemical composition.

Characterization of sound production by the pot-bellied seahorse (Hippocampus abdominalis) during feeding.

Sound production during feeding by the pot-bellied seahorse, Hippocampus abdominalis, was quantified with an observation of clicks (acoustic signal) and snicks (visual behavior). Female, male, and juvenile seahorses had feeding sounds characterized for peak (dominant) frequency (Hz), sound pressure level (SPL), and duration (ms). Subject body size and condition was estimated by standard length (SL, cm), to determine an estimate of body condition index (BCI). An inverse correlation between mean peak frequency (Hz) of clicks and SL was found for females. A negative correlation between peak frequency (Hz) of clicks and a residual BCI was determined for both males and females, suggesting that acoustic signals may contain information regarding fitness.

Development of gonad and brood pouch and expression profiles of cgα, fshβ and lhβ transcripts and plasma 17β-E2 and 11-KT during the first reproductive cycle of farmed big-belly seahorse (Hippocampus abdominalis)

Limited data are available regarding the gonadal development of farmed seahorse along with the physiological role of gonadotropins and sex steroids during gonadal maturation. In the present study, we conducted an examination of the developmental stages of gonad and brood pouch during the initial reproductive cycle of the farmed big-belly seahorse (Hippocampus abdominalis), and our results showed that males sexually matured half of a month later than females, and the brood pouch emerged at 3.5-month old and then reached the late-stage at 5.5-month old. Meanwhile, the open reading frame (ORF) of gonadotropin subunits (cgα, fshβ and lhβ) of the big-belly seahorse were cloned and characterized, and tissue distribution profiles of their transcripts were analyzed, revealing their detectability across various tissues, with a notable presence observed in the brain tissues. Furthermore, expression profiles of cgα, fshβ and lhβ transcripts and the plasma levels of 17β-estradiol (E2) and 11-ketotestosterone (KT) in farmed big-belly seahorse during the first reproductive cycle were also investigated. The findings indicated that cgα, fshβ and lhβ transcripts exhibited low expressions at the initial development of ovary or testis, raised continuously and reached their peak levels at the period of spawning or spermation, and they kept gradually increasing accompany with brood pouch growth. It is noteworthy that variation of mRNA levels of fshβ were slightly higher than those of lhβ. Besides, the results showed that plasma 11-KT levels presented an increasing trend during testicular development, peaking in fish with III and IV-stage testis and early-stage brood pouch, and plasma 17β-E2 levels were higher in vitellogenic stage of fish with II-stage ovary and early III-stage ovary. Taken together, these data reveal that gonadotropins and sex steroids likely play a role in the regulation of gonad and brood pouch growth in seahorse. These observations not only contribute to clarifying function of reproductive hormones on growth of gonad and brood pouch of seahorse, but also providing an important theoretical foundation for the artificial breeding of seahorses.

Paternal protein provisioning to embryos during male seahorse pregnancy.

Seahorses exhibit male pregnancy and are thus valuable comparative models for the study of the physiology and evolution of pregnancy. This study shows that protein is transported from fathers to developing embryos during gestation, and provides new knowledge about paternal contributions to embryonic development. Syngnathid embryos (seahorses, pipefishes and seadragons) develop on or in the male in a specialised brooding structure (brood pouch). Seahorse brood pouches supply nutrients, including lipids, to developing embryos (patrotrophy). We tested the hypothesis that proteins, vital for gene regulation and tissue growth during embryogenesis, are also transported from father to embryos, using the Australian pot-bellied seahorse, Hippocampus abdominalis. We used dry masses and total nitrogen content to estimate the total protein content of newly fertilised egg and neonate H. abdominalis. Neonates contained significantly greater protein mass than newly fertilised eggs. This result indicates that paternal protein transport to developing embryos occurs during H. abdominalis pregnancy. This study is the first to show paternal protein transport during pregnancy in seahorses, and furthers our understanding of paternal influence on embryonic development in male pregnant vertebrates.

The Early Allometric Growth and Osteological Ontogeny of Pot-Bellied Seahorse (Hippocampus abdominalis, L. 1827) under Mass-Scale Captive Breeding Conditions in North China

Seahorses are valuable species for their use in traditional Chinese medicine, as well as for the aquarium trade as ornamentals and curiosities. To balance market demand and reduce pressure on wild populations, many countries have undertaken commercial seahorse cultivation. Skeletal development plays a crucial role in fish fry culture, affecting external morphology, feeding, and movement. This study investigated the ontogeny allometry, timing, and progression of skeletal development in H. abdominalis from DAB (day after birth) 1 to DAB 100 under mass-scale captive breeding conditions in north China. The results of this study revealed the growth rate was significantly increased between DAB 30 and DAB 54. Allometry analysis revealed that in the early stage, the head, trunk, and tail demonstrated almost isometric growth. However, in the later stage, the head and trunk exhibited negative isometric growth, whereas the tail displayed positive isometric growth. Skeletal staining results showed that newborn seahorses do not have ossified bones until DAB 11 (SL 28.14 ± 2.94 mm). Ossification was primarily observed in the jaw region and the tubular nasal structure of the cranium, which indicated the importance of the early development of feeding organs. The initial formation of ossified vertebral columns was observed at DAB 13 (SL 26.48 ± 0.63 mm), with the complete ossification of all vertebrae occurring by DAB 45 (SL 54.87 ± 4.70 mm). Furthermore, the cranium, rings, and plates were all fully ossified by DAB 30. Ossification of the fins began at DAB 23 (SL 31.27 ± 4.05 mm). However, neither of them were fully ossified by DAB 100. The pelvic fin and the complete structure of the caudal fin were not observed, possibly because of caudal fin ray structure degeneration within the pouch. In addition, no skeletal deformities were observed in all the tested samples. The results of this study provide valuable information on the developmental biology of H. abdominalis, enriching our understanding of their growth and offering insights for optimizing fish fry breeding technologies.

Gonad and Germ Cell Development and Maturation Characteristics of the Pot-Bellied Seahorse (Hippocampus abdominalis) under Captive Breeding Conditions in Northern China

Ovoviviparity and male pregnancy represent distinctive reproductive strategies in seahorses. However, the detailed process of gonadal development in seahorses, particularly in the pot-bellied seahorse (Hippocampus abdominalis), remains largely unknown. In this study, we investigated the complete gonadal development process of the pot-bellied seahorse under captive breeding conditions (18 ± 1 °C). Immediately after birth, primordial germ cells (PGCs) were found within the genital ridge, enclosed by a single layer of somatic cells. Around 7–9 days after birth (DAB), the ovary begins to differentiate. By 30 DAB, two germinal ridges had formed along the edge of the follicular lamina in the ovary. The primary oocytes, resulting from this differentiation process, gradually migrated from the dorsal sides to the mid-ventral area of the ovary, eventually maturing into eggs. In the testis, the primary and secondary spermatocytes appeared at 15 and 30 DAB, respectively, preceding the formation of the testicular lumen (50 DAB). The testis was observed to consist of a single large germinal compartment. Under captive breeding conditions in Northern China, the pot-bellied seahorse demonstrated year-round breeding capability, with each male producing approximately 100–150 larvae. The findings from this study contribute valuable insights into seahorse aquaculture and enhance understanding of the unique reproductive strategy employed by seahorses.

Identification, expression profiling, and functional characterization of cystatin C from big-belly seahorse (Hippocampus abdominalis).

Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. Emerging evidence suggests the immune regulatory roles of CSTC in antigen presentation, the release of different inflammatory mediators, and apoptosis in various pathophysiologies. In this study, the 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse (Hippocampus abdominalis) was cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in ovaries. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 14.29-kDa protein of recombinant HaCSTC (rHaCSTC) was expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, HaCSTC overexpression strongly decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability. Our findings imply the profound role of HaCSTC against pathogen infections by modulating fish immune responses.

Orphan gene expressed in flame cone cells uniquely found in seahorse epithelium.

The seahorse is one of the most unique teleost fishes in its morphology. The body is surrounded by bony plates and spines, and the male fish possess a brooding organ, called the brood pouch, on their tail. The surfaces of the brood pouch and the spines are surrounded by characteristic so-called flame cone cells. Based on our histological observations, flame cone cells are present in the seahorse Hippocampus abdominalis, but not in the barbed pipefish Urocampus nanus or the seaweed pipefish Syngnathus schlegeli, both of which belong to the same family as the seahorse. In the flame cone cells, we observed expression of an "orphan gene" lacking homologs in other lineages. This gene, which we named the proline-glycine rich (pgrich) gene, codes for an amino acid sequence composed of repetitive units. In situ hybridization and immunohistochemical analyses detected pgrich-positive signals from the flame cone cells. Based on a survey of the genome sequences of 15 teleost species, the pgrich gene is only found from some species of Syngnathiformes (namely, the genera Syngnathus and Hippocampus). The amino acid sequence of the seahorse PGrich is somewhat similar to the sequence deduced from the antisense strand of elastin. Furthermore, there are many transposable elements around the pgrich gene. These results suggest that the pgrich gene may have originated from the elastin gene with the involvement of transposable elements and obtained its novel function in the flame cone cells during the evolution of the seahorse.

Edwardsiella piscicida infection reshapes the intestinal microbiome and metabolome of big-belly seahorses: mechanistic insights of synergistic actions of virulence factors.

Uncovering the mechanism underlying the pathogenesis of Edwardsiella piscicida-induced enteritis is essential for global aquaculture. In the present study, we identified E. piscicida as a lethal pathogen of the big-belly seahorse (Hippocampus abdominalis) and revealed its pathogenic pattern and characteristics by updating our established bacterial enteritis model and evaluation system. Conjoint analysis of metagenomic and metabolomic data showed that 15 core virulence factors could mutually coordinate the remodeling of intestinal microorganisms and host metabolism and induce enteritis in the big-belly seahorse. Specifically, the Flagella, Type IV pili, and Lap could significantly increase the activities of the representative functional pathways of both flagella assembly and bacterial chemotaxis in the intestinal microbiota (P < 0.01) to promote pathogen motility, adherence, and invasion. Legiobactin, IraAB, and Hpt could increase ABC transporter activity (P < 0.01) to compete for host nutrition and promote self-replication. Capsule1, HP-NAP, and FarAB could help the pathogen to avoid phagocytosis. Upon entering epithelial cells and phagocytes, Bsa T3SS and Dot/Icm could significantly increase bacterial secretion system activity (P < 0.01) to promote the intracellular survival and replication of the pathogen and the subsequent invasion of the neighboring tissues. Finally, LPS3 could significantly increase lipopolysaccharide biosynthesis (P < 0.01) to release toxins and kill the host. Throughout the pathogenic process, BopD, PhoP, and BfmRS significantly activated the two-component system (P < 0.01) to coordinate with other VFs to promote deep invasion. In addition, the levels of seven key metabolic biomarkers, Taurine, L-Proline, Uridine, L-Glutamate, Glutathione, Xanthosine, and L-Malic acid, significantly decreased (P < 0.01), and they can be used for characterizing E. piscicida infection. Overall, the present study systematically revealed how a combination of virulence factors mediate E. piscicida-induced enteritis in fish for the first time, providing a theoretical reference for preventing and controlling this disease in the aquaculture of seahorses and other fishes.

Supplementation with Enterococcus faecium enhances growth performance, intestinal health and immunity of big-belly seahorses (Hippocampus abdominalis) during diet conversion

Diet conversion is a key stage of seahorse aquaculture. Herein, we set up five groups to evaluate the probiotic effects of Enterococcus faecium supplementation on the growth, development, intestinal health, and immunity of big-belly seahorses (Hippocampus abdominalis) during this stage, including control (C) (no E. faecium), H2 (1 ×107 CFU mL−1, every 2 d), H4 (1 ×107 CFU mL−1, every 4 d), L2 (1 ×105 CFU mL−1, every 2 d), and L4 (1 × 105 CFU mL−1, every 4 d) groups. After 48 d culture, concentration and frequency of E. faecium supplementation, as well as their interaction, were confirmed to have significant effects on growth performance of seahorses (P < 0.05). Body weight, specific growth rate, body weight increase, standard length and standard length increase of H4 and L4 groups were significantly higher (P < 0.05); Ovary weight but not testis weight and brood pouch length significantly increased in H4 group (P < 0.05); Feed intake increased (P < 0.05), while feed conversion ratio significantly reduced in H4 group (P < 0.05); Together with the increase of intestinal absorption area (P < 0.05) and digestive enzymes in H4 group, it suggests that the increased growth performance of big-belly seahorses may be attributed to the integrated improvements in food intake, digestion, and absorption capacity. Additionally, significant increase of expression of intestinal immune-related genes, and remodeling of both intestinal microbiota and their functions endowed seahorses with healthier intestinal environment, which resulted in the increase of survival rate by 50% against pathogenic Edwardsiella tarda challenge. Constructing comprehensive evaluation systems of both growth performance and probiotic effects for the first time could help us determine the best application strategy of E. faecium during the diet conversion period of big-belly seahorse aquaculture. This finding may provide a novel reference for the application of E. faecium in seahorse aquaculture.

Antioxidant potential of hydrolysate-derived seahorse (Hippocampus abdominalis) peptide: Protective effects against AAPH-induced oxidative damage in vitro and in vivo

In this study, seahorse peptide (SHP) was isolated from an alcalase-treated hydrolysate from Hippocampus abdominalis and assessed for its antioxidant potential against AAPH-induced oxidative stress damage. AAPH stimulation significantly decreased cell viability and increased intracellular reactive oxygen species (ROS) production in Vero cells. SHP treatment increased cell viability and remarkably lowered ROS production under AAPH-induced oxidative stress. Furthermore, it protected against AAPH-induced apoptotic DNA damage. Western blot analysis demonstrated that SHP treatment remarkably increased the protein expression levels of catalase and SOD in AAPH-induced Vero cells. A zebrafish study revealed that SHP-treated zebrafish embryos resulted in lower cell death, ROS generation, and lipid peroxidation than the AAPH-treated group. These results suggest that SHP is a potent functional antioxidant that could be developed as a natural antioxidant in the food and functional food industries.

Establishment of preliminary reference intervals and cytochemical staining of blood cells in big-bellied seahorses (Hippocampus abdominalis).

Despite their popularity, hematology reference intervals (RIs) have not been established in big-bellied seahorses (Hippocampus abdominalis). The objectives of this study were to establish hematologic RIs to compare values between sex in regard to cytochemical staining of blood cells. We also sought to compare white blood cell concentrations using the Natt and Herrick technique vs blood smear estimates. Forty-three healthy individuals from the Aquarium du Québec (22 females and 21 males) were included. Normal health status was confirmed by an unremarkable physical examination in five individuals and by necropsy of five other individuals, of which all were excluded from further analyses. Venipuncture was performed from the ventral coccygeal vein in the remaining 33 individuals without anesthesia using heparinized insulin syringes. A blood volume of 0.05 to 0.1ml was collected to prepare Wright Giemsa-stained blood smears and hematocrits immediately after venipuncture. Whole blood was stored in heparinized Eppendorf tubes to determine red and white blood cell concentrations using the Natt and Herrick technique with a hemocytometer in 10 individuals; these results were compared with blood smear estimates. Additional blood smears were stained with alkaline phosphatase substrate, periodic acid Schiff, and toluidine blue stains. The reference intervals included the packed cell volume (27.4-67.5%), thrombocyte count (19.5-197.7 × 109 /L), and white blood cell (WBC) count (2-54.8 × 109 /L), including neutrophils (1.1-21.3 × 109 /L), lymphocytes (2.7-45.5 × 109 /L), and monocytes (0-2.2 × 109 /L). The WBC hemocytometer counts showed no correlation with blood smear estimates (Spearman's rho=0.2). There was also no significant difference between the sexes. These preliminary reference intervals will help assess the health of seahorses.

Seahorse brood pouch morphology and control of male parturition in Hippocampus abdominalis

IntroductionSyngnathids (seahorses, pipefishes and seadragons) are among the few vertebrates that display male pregnancy. During seahorse pregnancy, males incubate developing embryos embedded in a placenta within a fleshy brood pouch, before expelling fully developed neonates at parturition. The mechanisms underpinning seahorse parturition are poorly understood. MethodsWe examined the morphology of the brood pouch using microcomputed tomography and histological techniques, in combination with physiological assays, to examine how male pot-bellied seahorses (Hippocampus abdominalis) control labour. In female-pregnant vertebrates, nonapeptide hormones (such as vasopressin- and oxytocin-like hormones) produce contractions of gestational smooth muscle to produce labour. ResultsHistological analysis of the seahorse brood pouch reveals only scattered small smooth muscle bundles in the brood pouch, and in-vitro application of isotocin (a teleost nonapeptide hormone) to the brood pouch do not produce measurable muscle contractions. Micro-computed tomography shows differences in size and orientation of the anal fin assembly between male and female pot-bellied seahorses, and histological analysis reveals large skeletal muscle bundles attached to the anal fin bones at the male brood pouch opening. DiscussionWe conclude that seahorse parturition may be facilitated by contraction of these muscles, which, in combination with body movements, serves to gape open the pouch and expel the neonates. Future biomechanical studies are needed to test this hypothesis.

Isolation and identification of a new strain of nervous necrosis virus from the big-belly seahorse Hippocampus abdominalis

BackgroundBetanodaviruses, members of the Nodaviridae family, are the causative agents of viral nervous necrosis in fish, resulting in great economic losses worldwide.MethodsIn this study, we isolated a virus strain named seahorse nervous necrosis virus (SHNNV) from cultured big-belly seahorses Hippocampus abdominalis in Xiamen city, Fujian Province, China. Virus isolation, PCR detection, phylogenetic analysis, qRT-PCR, fluorescence in situ hybridization and histology were used for virus identification and analysis of virus histopathology. Furthermore, an artificial infection experiment was conducted for virulence testing.ResultsBrain and eye tissue homogenates of diseased big-belly seahorses were inoculated onto a grouper spleen (GS) cell monolayer at 28 °C. Tissue homogenates induced obvious cytopathic effects in GS cells. PCR and sequencing analyses revealed that the virus belonged to Betanodavirus and shared high sequence identity with red-spotted grouper nervous necrosis virus isolates. qRT-PCR and fluorescence in situ hybridization revealed that SHNNV mainly attacked the brain and eye. Histopathological examination revealed that the virus led to cytoplasmic vacuolation in the brain and retinal tissues. Infection experiments confirmed that SHNNV was highly infectious, causing massive death in big-belly seahorses.ConclusionA novel seahorse betanodavirus from the big-belly seahorse cultured in China was discovered. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

Antihypertensive Effects of IGTGIPGIW Peptide Purified from Hippocampus abdominalis: p-eNOS and p-AKT Stimulation in EA.hy926 Cells and Lowering of Blood Pressure in SHR Model.

The aim of this study was to assess the potential hypertensive effects of the IGTGIPGIW peptide purified from Hippocampus abdominalis alcalase hydrolysate (HA) for application in the functional food industry. We investigated the antihypertensive effects of IGTGIPGIW in vitro by assessing nitric oxide production in EA.hy926 endothelial cells, which is a major factor affecting vasorelaxation. The potential vasorelaxation effect was evaluated using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, a fluorescent stain. IGTGIPGIW significantly increased the expression of endothelial-derived relaxing factors, including endothelial nitric oxide synthase and protein kinase B, in EA.hy926 cells. Furthermore, oral administration of IGTGIPGIW significantly lowered the systolic blood pressure (183.60 ± 1.34 mmHg) and rapidly recovered the diastolic blood pressure (143.50 ± 5.55 mmHg) in the spontaneously hypertensive rat model in vivo. Our results demonstrate the antihypertensive activity of the IGTGIPGIW peptide purified from H. abdominalis and indicate its suitability for application in the functional food industry.

Molecular characterization, expression, and functional analysis of cystatin B in the big-belly seahorse (Hippocampus abdominalis)

Cystatins are a diverse group of cysteine protease inhibitors widely present among various organisms. Beyond their protease inhibitor function, cystatins play a crucial role in diverse pathophysiological conditions in animals, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. However, the role of cystatins in immunity against viral and bacterial infections in fish remains to be elucidated. In this study, the cystatin B from big-belly seahorse, Hippocampus abdominalis, designated as HaCSTB, was identified and characterized. HaCSTB shared the highest homology with type 1 cystatin family members of teleosts and had three cystatin catalytic domains with no signal peptides or disulfide bonds. HaCSTB transcripts were mainly expressed in peripheral blood cells (PBCs), followed by the testis and pouch of healthy big-belly seahorses. Immune challenge with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (Poly I:C), and Streptococcus iniae induced upregulation of relative HaCSTB mRNA expression in PBCs. Subcellular localization analysis revealed the distribution of HaCSTB in the cytosol, mitochondria, and nuclei of fathead minnow cells (FHM). Recombinant HaCSTB (rHaCSTB) exhibited potent in vitro inhibitory activity against papain, a cysteine protease, in a concentration-, pH-, and temperature-dependent manner. Overexpression of HaCSTB in viral hemorrhagic septicemia virus (VHSV)-susceptible FHM cells increased cell viability and reduced VHSV-induced apoptosis. Collectively, these results suggest that HaCSTB might engage in the teleostean immune protection against bacteria and viruses.