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Tributyltin (TBT), an antifouling biocide frequently detected in aquatic systems, is generally considered to be an environmental obesogen. However, alterations in lipid metabolism in aquatic animals that are exposed to TBT are scarcely known. This study examined the effects of in vitro exposure to TBT on hepatic lipid homeostasis in the lined seahorse (Hippocampus erectus). Primary seahorse hepatocyte cultures were established for the first time. TBT exposure (100 and 500 nM for 24 h) significantly promoted lipid accumulation in seahorse hepatocytes and drastically reduced the number of active intracellular lysosomes. Furthermore, exposure to TBT significantly upregulated the gene expression of lipogenic enzymes and transcription factors but downregulated that of genes involved in the catabolism of lipid droplets in seahorse hepatocytes. These results indicate that TBT disrupts hepatic lipid homeostasis by simultaneously promoting lipid synthesis and inhibiting lipid droplet breakdown in seahorses. The present study extends our understanding of the utilization of primary hepatocytes from marine animals for toxicological research, and the molecular evidence of the effects of TBT on hepatic lipid homeostasis in teleost fishes. [Display omitted] • Seahorse primary hepatocyte cultures were established for the first time. • TBT-induced hepatic lipid accumulation was related to exposure concentrations. • Lipid droplets increased while lysosomes decreased in TBT-exposed hepatocytes. • TBT altered the expression of lipid metabolism genes in seahorse hepatocytes. [ABSTRACT FROM AUTHOR]

Considering that the intestine is always in close contact with microorganisms and is vulnerable to pathogen invasion (Lawley & Walker, 2013; Li et al., 2015), the highest expression pattern implied that the two seahorse sPLA2-XIIBs might play an important role in intestinal immunity. Keywords: antibacterial response; intestine; seahorse; secretory phospholipase A2 group XIIB EN antibacterial response intestine seahorse secretory phospholipase A2 group XIIB 1059 1063 5 06/10/22 20220701 NES 220701 Aquatic diseases caused by pathogenic bacteria have seriously hindered the development of seahorse aquaculture, which is supposed to bridge the gap between the increasing demand for medicinal markets and decreasing wild seahorse populations (Jiang et al., 2020; Li et al., 2020; Qin et al., 2017). Therefore, the two sPLA2-XIIBs identified in seahorses are homologous to the sPLA2-XIIBs in teleosts. The qPCR results showed that the expression levels of the two sPLA2-XIIB genes in the intestine of the I Vibrio i -infected seahorses were significantly higher than those of healthy seahorses (Figure 3a). [Extracted from the article]

• The sequences of seahorse LPXRFa-1 and LPXRFa-2 are unique among vertebrates. • The synteny blocks of GnIH gene loci are conserved among the vertebrates. • The seahorse GnIH mRNA expressed highly in the hypothalamus and brood pouch. • The GnIH mRNA expression reduced significantly at the early puberty stage of the male seahorse. • The GnIH gene was up-regulated at the pregnancy stage of the male seahorse. The gonadotropin-inhibitory hormone (GnIH) plays a negative role in the hypothalamic-pituitary-gonadal (HPG) axis by inhibiting gonadotropin secretion in vertebrates. Male pregnancy and ovoviviparous behavior are unique phenomena among vertebrates. To better understand the neuroendocrine regulatory mechanisms in ovoviviparous fish with male pregnancy, we identified the orthologous GnIH gene in the lined seahorse (Hippocampus erectus). The full-length cDNA of the GnIH precursor was 658 base pairs with an open reading frame of 528 base pairs that encoded a 175-amino acid prepro-GnIH peptide. The seahorse GnIH precursor contained two putative LPXRFamide peptides. Both seahorse LPXRFa-1 and LPXRFa-2 were found to be unique among vertebrates. The synteny blocks of GnIH gene loci were conserved in mammals and teleosts. Tissue distribution analysis revealed that seahorse GnIH mRNA was mainly expressed in the hypothalamus, with relatively high levels observed in the brood pouch. The expression patterns of seahorse GnIH during different reproductive stages and pregnancy stages were also detected, and GnIH mRNA expression was significantly reduced during the early puberty stage. In addition, GnIH mRNA expression was significantly increased during the pregnancy stage compared to non-pregnancy stages. In summary, our results reveal the existence of GnIH in ovoviviparous fish and suggest its involvement in regulation of reproductive behavior and male pregnancy in the male seahorse. [ABSTRACT FROM AUTHOR]

Background: The evolution of male pregnancy is the most distinctive characteristic of syngnathids, and their specialized life history traits make syngnathid species excellent model species for many issues in biological evolution. However, the origin of syngnathids and the evolutionary divergence time of different syngnathid species remain poorly resolved. Comprehensive phylogenetic studies of the Syngnathidae will provide critical evidence to elucidate their origin, evolution, and dispersal patterns. Results: We sequenced the mitochondrial genomes of eight syngnathid species in this study, and the estimated divergence times suggested that syngnathids diverged from other teleosts approximately 48.8 Mya during the Eocene period. Selection analysis showed that many mitochondrial genes of syngnathids exhibited significantly lower Ka/Ks values than those of other teleosts. The two most frequently used codons in syngnathid fishes were different from those in other teleosts, and a greater proportion of the mitochondrial simple sequence repeats (SSRs) were distributed in non-coding sequences in syngnathids compared with other teleosts. Conclusions: Our study indicated that syngnathid fishes experienced an adaptive radiation process during the early explosion of species. Syngnathid mitochondrial OXPHOS genes appear to exhibit depressed Ka/Ks ratios compared with those of other teleosts, and this may suggest that their mitogenomes have experienced strong selective constraints to eliminate deleterious mutations. [ABSTRACT FROM AUTHOR]

Seahorse is characterized by its male pregnancy and sex-role reversal. To better understand the sexual dimorphism of male and female seahorses based on essential genes, we performed systematic transcriptome studies for both genders. A total of 157,834,590 cleaned reads were obtained and assembled into 129,268 transcripts and 31,764 could be annotated. Results showed that 176 up-regulated and 391 down-regulated transcripts were identified in the male seahorses compared with those in females. Genes involved in sex differentiation, such as dmrt1, sox9, fem1 and vasa , were identified and characterized. Moreover, the essential genes involved in reproductive molecular pathway were identified and analyzed in seahorses. In conclusion, the present study provides an archive for the future systematic research on seahorse sex differentiation. • Sexual dimorphism of male and female seahorses was studied through transcriptome comparison. • 176 up-regulated transcripts were identified in male seahorses. • Genes involved in sex differentiation, such as dmrt1, sox9 and fem1 , were characterized. [ABSTRACT FROM AUTHOR]

The unique body morphology and specialized life history traits make seahorses excellent flagship species for many issues in marine conservation and biological evolution. In this study, the complete mitochondrial genome of Hippocampus fuscus was determined. Phylogenetic analyses showed that H. fuscus had a close genetic relationship to Hippocampus reidi, which give us a new insight into the speciation and dispersal among seahorse genus. A total of 843 base pairs of cytochrome b (Cytb) gene and a 646 base pairs of cytochrome oxidase I (COI) gene were obtained from 36 H. fuscus and 26 H. reidi from three populations. Population genetic analysis revealed a relatively high genetic diversity across the populations of H. fuscus compared with H. reidi. Neighbour-joining (NJ) tree of COI and Cytb gene sequences showed that H. fuscus haplotypes formed one cluster. The result of median-joining network of haplotypes based on COI and Cytb indicated a lack of structure in populations of H. fuscus. This study addressed the information about the evolution history and genetic structure of an Arabian seahorse H. fuscus, which provides insight into the genetic conservation and sustainable management for this species. [ABSTRACT FROM AUTHOR]

Seahorses are one of the most amazing ovoviviparous fishes in the ocean because males, and not females, have evolved a brood pouch for incubating embryos. During male pregnancy, paternal seahorses need to develop effective immune protection for embryos in the brood pouch from potential infection by pathogens. Lysozymes (Lyz) are a group of antibacterial enzymes of the innate immune system that play an important role in resisting pathogen invasion. However, the immune function of lysozymes in the brood pouch of the pregnancy-lined seahorse (Hippocampus erectus) remains unknown. In this study, we found three different lysozymes in the lined seahorse: HeLyzC , HeLyzG1 , and HeLyzG2. Synteny analysis revealed that HeLyzG1 and HeLyzG2 were generated by species-specific expansion rather than tandem duplication. Tissue expression patterns showed that the highest mRNA expression levels of the three lysozymes occurred in the brood pouches. Immunostimulation-induced expression analysis showed that all three HeLyzs in the brood pouches up-regulated their mRNA expression levels after Vibrio parahaemolyticus infection, but only the HeLyzG2 was upregulated after Poly(I:C) injection. Similarly, except for HeLyzC , upregulated expressions of HeLyzG1 and HeLyzG2 were found quickly in brood pouches injected with LPS. The upregulated levels of HeLyzC and HeLyzG2 in brood pouches during pregnancy were significantly higher than those in non-pregnancy, implying that seahorse lysozymes might function in the immune defense in brood pouches during pregnancy. In addition, the expression levels of HeLyzs were low in embryos in the brood pouch but significantly increased in neonates. This implies that embryos in the brood pouch might not necessarily express more lysozymes by themselves due to paternal immune protection. In conclusion, our study demonstrated that HeLyzs play an important role in immune protection during male seahorse gestation, and the synergistic effect of multiple HeLyzs may contribute to improved neonatal survival. • Three isoforms of seahorse lysozyme were highly expressed in the brood pouch. • Immunostimulation induced up-regulations of lysozymes proved defense against pathogen. • Higher expressions in pregnant brood pouch indicated functions in male pregnancy. • Low expressions in embryos suggested dependence on paternal immune protection. [ABSTRACT FROM AUTHOR]

DNA barcoding offers a rapid and accurate assessment for species labelling and identification. Here, we investigated the performance of DNA barcodes in a sample of Syngnathidae, a unique group of fish with male pregnancy. A total of 1002 DNA barcodes using 649 base-pair fragments of the cytochrome c oxidase subunit I (COI) gene were generated. All species were associated with distinct DNA barcode and could be readily distinguished; seven of the COI barcode clusters represented the first species records submitted to the Barcode of Life Data Systems (BOLD) and GenBank databases. In the Neighbor-joining tree of COI sequences, two major clusters (Gastrophori and Urophori) were displayed, which could also be classified by their brood pouches. However, the tail-brooding Hippichthys cyanospilus and trunk-brooding Syngnathoides biaculeatus were reverse-clustered together with the Gastrophori and the Urophori, respectively. Both seahorse and pipefish sequences showed high frequencies of nucleotide substitutions. The probability of nucleotide substitutions, those in pipefish were higher than that of all seahorses. However, we identified no signal for positive selection based on the COI gene in any of the data sets. Our results supported DNA barcoding as an efficient molecular tool for achieving better monitoring, conservation, and management of fisheries. [ABSTRACT FROM AUTHOR]

Seahorses (genus Hippocampus ) are flagship species for marine conservation and evolutionary studies. Wild seahorse populations have declined sharply because of overfishing or other disrupting activities, which are attracting increasing global concern for their threatened status. For study of their population status, the identification of young seahorses by morphological examination has been controversial and remains a difficult task due to overlapping of meristic and morphological characters. Here we reported a simple and sensitive method to identify seahorse species from China based on cross-species microsatellites. The cross-species amplification success rate varied from 40.0% to 90.0% depending on the species, with a mean value of 59.6%. According to allele presence/absence of cross-species microsatellites, all the individuals representing 10 seahorse species could be accurately identified using one or more loci. In addition, we found both genome size and genetic distance between the source and target species negatively correlated with microsatellite cross-species amplification success. Moreover, a Neighbor-Joining dendrogram was constructed based on genetic similarity and suggested that the 10 seahorse species could be divided into four groups. These results demonstrate our method could improve the identification of seahorses, providing data to understand seahorse stock structure and recruitment dynamics, and therefore resources conservation. [ABSTRACT FROM AUTHOR]

Seahorse is a specialized animal due to its male pregnant strategy in the brood pouch, which can evidently provide immune protection, nutrition, and osmoregulation to the offspring. Secretory phospholipase A2 group IB (sPLA2-IB), which encodes a secreted phospholipase A2 that functions for metabolic regulation and antibacterial defense, plays a vital role for immune regulation in teleost, but its potential immune functions in seahorse remain unknown. Here, we aimed to evaluate the characteristics and functional profiles of sPLA2-IBs in lined seahorse (Hippocampus erectus), which is one of the most important breeding seahorse species in the world. Results showed that most teleost species have only one sPLA2-IB gene, while the seahorses presented two tandem repetitive copies (sPLA2-IBa and sPLA2-IBb). This finding showed that the highest expression levels of sPLA2-IBs in seahorses were in the liver, and significant up-regulation could be induced by lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly[I:C]), as well as the seahorse's common pathogen Vibrio parahaemolyticus. Interestingly, the expression levels of sPLA2-IBs in the brood pouch showed an upward trend during pregnancy, compared with that in non-pregnancy stage, and peaked in the late pregnancy stage, implying that seahorse sPLA2-IBs in brood pouch might function for immune defense during pregnancy. In addition, seahorse sPLA2-IBa was significantly highly expressed in early developing embryos in brood pouch, but sPLA2-IBb expression dramatically increased in new-born juveniles, which implies the functional divergence between the two sPLA2-IBs by different ontogenetic expression. In conclusion, this study indicated that seahorse sPLA2-IBs play an important role in the immunoprotection during male pregnancy, and functional co-option of seahorse sPLA2-IBs might be conducive to improving the survival rate of its offspring. • Seahorses presented two lineage-specific tandem repetitive sPLA2-IB genes. • Challenge induced up-regulation proved their immune defense against pathogen. • Higher expressions during pregnancy suggest potential immune function in the brood pouch. [ABSTRACT FROM AUTHOR]

The innate immune system plays an important role in the immune responses of fish, particularly in immunodeficient seahorses, which are vulnerable to infection. In this study, we investigated the evolutionary adaptation of the Toll-like receptor 5 signaling in the lined seahorse (Hippocampus erectus) and the physiological and molecular responses of seahorse to bacterial and parasitic tapeworm infections. Four sites under positive selection among three genes, hsp70 , tlr5 , and tak1 , showed Bayes Empirical Bayes values >0.95. The respiratory rate of H. erectus increased after being challenged with Vibrio and Scalithrium. Stress-induced expression of hsp60 and hsp70 , which can bind PAMP molecules and modulate PAMP-induced Toll-like receptor (TLR) signaling, was significantly upregulated in immune organs such as the gill, intestine, kidney, and liver. Additionally, the MyD88-dependent TLR signaling pathway of H. erectus was activated in response to both bacterial and parasitic infections. The expression profiles of TLR signaling molecules revealed that these molecules mediate immune processes and act against various pathogens. These results improve our understanding of the functional role of key genes involved in PAMP-mediated signaling and provide theoretical support for the prevention and control of diseases in cultured seahorses. • Amino acid changes in PAMP-induced TLR signaling genes were positively selected. • The PAMP-triggered immunity pathway adaptively evolves in immunodeficient seahorse. • The MyD88-dependent TLR signaling molecules in H. erectus is upregulated against various pathogens during infection. [ABSTRACT FROM AUTHOR]

The seahorse is a typical suction feeder with an elongated, tubular snout and a small terminal mouth. It is important to determine the details of this unique prey-capture behavior to improve the survival rate during seahorse breeding, especially for juveniles. In this study, we described the sucking process and characterized the kinematics in juvenile lined seahorse (Hippocampus erectus) at different ages (1–30 days after parturition; DAP) using a high-speed video system, and also investigated the histological basis of the feeding-related organs, including snout, eye, and intestine. The sucking of juvenile seahorses can be divided into four steps: searching, locating, capturing, and swallowing. Seahorses capture prey mainly based on the vision system, and histological results revealed a developed visual acuity in the newborn seahorses, so that they were able to prey once after releasing from the male seahorse's brood pouch. The attack distance, maximum snout width, capture success rate, and daily food intake of juvenile seahorses increased with maturation. A remarkable inflection point, in the snout expansion rate, successful predation rate, and growth rate, occurs at about 10 DAP, when calcification of the snout bones was completed. In addition, the intestines of newborn seahorses were short and quite simple, but developed rapidly during the first 20 DAP. These findings provide a better understanding of prey capture kinematics and nutritional physiology, and will be beneficial for improving the feeding strategies for juveniles during large seahorse breeding. • Sucking kinematics in juvenile seahorses at different ages was described. • Developed visual acuity was observed in the newborn seahorses. • Snout calcification and intestines development affect juvenile's nutrition intake. [ABSTRACT FROM AUTHOR]

The male pregnancy of seahorses is unique, but their reproductive response to environmental disturbances has not yet been clarified. Tributyltin (TBT) is known to have an endocrine disrupting effect on the reproductive system of coastal marine organisms. This study evaluated the potential effects of exposure to environmentally relevant concentrations of TBT on the development of gonads and brood pouch of the lined seahorse (Hippocampus erectus). Physiological, histological, and transcriptional analyses were conducted, and results showed that high levels of TBT bioaccumulation occurred in male and female seahorses. TBT led to ovarian follicular atresia and apoptosis with the elevation of androgen levels, accompanied by the induction of genes associated with lysosomes and autophagosomes. Comparative transcriptional analyses revealed the likely inhibition of spermatogenesis via the suppression of cyclic AMP and androgen synthesis. Notably, the transcriptional profiles showed that TBT potentially affects the immune system, angiogenesis, and embryo nourishment of the brood pouch, which indicates that it has negative effects on the male reproductive system of seahorses. In summary, this study reveals that environmental levels of TBT potentially affect the reproductive efficiency of seahorses, and may ultimately lead to a reduction in their populations in coastal environments. ga1 • Tributyltin (TBT) led to significant follicular atresia in seahorse ovary. • Apoptosis, lysosomes and autophagosomes contributed to atresia in ovary. • TBT impaired spermatogenesis through regulating the androgen and cAMP synthesis. • TBT affected immunity, angiogenesis, and embryo nourishment of the brood pouch. [ABSTRACT FROM AUTHOR]

Seahorses (family Syngnathidae) are ovoviviparous fish whose embryos can obtain paternal immunoprotection through the male's brood pouch, which is a specialized organ for pregnancy. Previous studies have revealed that seahorses have a unique immune system and alpha-2 macroglobulin (α2M) may be involved in male pregnancy. To ascertain the significance of α2M in the immune system of the brood pouch, the characteristics of the α2M gene were first investigated in the lined seahorse (Hippocampus erectus). In this study, α2M showed a broad expression pattern, with the highest expression in the liver and brood pouch. Interestingly, during the six-month seahorse culture, expression level of α2M in the liver was particularly decreased during brood pouch formation (fourth and fifth months), but was restored the complete development of this brood pouch (sixth months), and a similar expression occurred in the brood pouch. This suggests that the seahorse might be able to regulate its immune response while a new organ develops. Further investigation also examined the immune regulation of α2M in brood pouches by injecting lipopolysaccharide and polyinosinic-polycytidylic acid. The results showed that, after the challenge experiment, α2M expression in the inner pseudoplacenta was upregulated, along with other important immune factors, such as Toll-like receptors (TLRs), tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6) and interferon-gamma (IFNγ). Taken together, our results reveal that α2M plays an important role in immunoprotection of the brood pouch, and may provide new insights into studies of male pregnancy regulation in seahorses. • α2M predominantly expressed in liver and brood pouch in seahorses. • α2M expression in the liver fluctuated, particularly during brood pouch formation. • LPS or Poly(I:C) up-regulated α2M mRNA level in the inner pseudoplacenta. • α2M is involved in the immunoprotection of the brood pouch in seahorses. [ABSTRACT FROM AUTHOR]

Seahorses, with brood pouch in adult males, are a bioindicator species that exhibit specialized reproductive strategy of "male pregnancy". Bisphenol A (BPA), one of the most pervasive endocrine-disrupting chemicals (EDCs), is hazardous for reproductive, immune, and neurological systems. However, no evidence of BPA toxicity to the male-pregnant animals is available. Herein, the reproductive toxicity of BPA was evaluated in lined seahorses (Hippocampus erectus) following exposure to environmentally relevant concentrations (10, 100, and 1000 μg/L) through physiological, histological, and transcriptional analyses. Our results indicated BPA bioaccumulation to be positively correlated with exposure doses in both sexes. Ovarian failure was only observed in the high-dose BPA treatment group, accompanied by the apoptosis of follicular cells and up-regulation of pro-apoptotic genes. However, brood pouches maintenance were surprisingly inhibited at low concentration, and transcriptomic analysis revealed disturbed profiles of genes involved in the extracellular matrix and cell-cell adhesion pathways. Interestingly, seahorse testes were less sensitive to BPA exposure than that in other teleosts. Thus, our study suggests that BPA at environmentally relevant concentrations might cause reproductive dysfunction in seahorses, potentially exerting adverse effects on the seahorse population since most of them inhabit shallow coastal areas with prevalent estrogenic contaminants. Unlabelled Image • BPA bioaccumulation was positively correlated with the exposure doses • BPA induced ovarian apoptosis by impacting apoptosis-related genes • Seahorse testis was less sensitive to BPA exposure than that of other teleost • BPA inhibited brood pouches maintenance via ECM and cell-cell adhesion genes [ABSTRACT FROM AUTHOR]

Exposure to environmental estrogens and progestins has contributed to adverse effects on the reproduction of many aquatic wildlife species. However, few reports have paid attention to fish species with specialized reproductive strategies, such as male-pregnancy seahorses. In this study, the potential effects on the behavior, gonad and brood pouch development, and transcriptomic profiles of lined seahorse Hippocampus erectus exposed to environmentally relevant concentrations of 17α-ethynyl estradiol (EE2, 5 ng/L, 50 ng/L, 10 ng/L, 100 ng/L) or progesterone (P4) for 60 days were examined. Both EE2 and P4 significantly inhibited male brood pouch development by disrupting the extracellular matrix and basement membrane pathways. In addition, both EE2 and P4 impaired the expression of genes associated with spermatogenesis in the testis, and even caused male feminization. We suggest that seahorses be regarded as a sensitive indicator for evaluating the potential effects of endocrine disrupting chemical (EDC) pollution on aquatic biotic communities. Unlabelled Image • Effects of EE2 and P4 on male-pregnancy seahorse are investigated. • Both EE2 and P4 improved ovary development at environmentally relevant concentrations. • Both EE2 and P4 inhibited male seahorse's brood pouch development. • Both EE2 and P4 down-regulated the transcription of spermatogenesis-related genes. [ABSTRACT FROM AUTHOR]