Male Seahorses Research Articles

Diversity and functional annotation of bacteria community associated with the epithelial surface of Hippocampus kuda (yellow seahorse) and its surrounding environment

Abstract. Maguate NJIFS, Tabugo SRM. 2024. Diversity and functional annotation of bacteria community associated with the epithelial surface of Hippocampus kuda (yellow seahorse) and its surrounding environment. Biodiversitas 25: 2230-2240. Hippocampus kuda, commonly known as the yellow seahorse, hosts various microorganisms crucial for its well-being and interactions within its ecosystem. This research aimed to analyze and understand the bacterial community associated with H. kuda by employing V3-V4 16S rRNA gene amplicon sequencing on the Illumina MiSeq platform. Four distinct libraries were constructed, representing the epithelial surfaces of both male and female seahorses and samples from water and soil/sediment habitats. Following rigorous quality control and processing, 187,972 Amplicon Sequence Variants (ASVs) were identified. The predominant ASVs were attributed to genera such as Vibrio, Roseobacter, Photobacterium, Ruegeria, Candidatus, Pseudoalteromonas, Synechococcus, Flavobacterium, and Altererythrobacter, along with some unidentified genera belonging to the Proteobacteria phylum. As indicated by the Shannon index, alpha diversity analysis demonstrated the highest bacterial diversity on the epithelial surface of male seahorses (MS), with a value of 3.2363. Moreover, the functional annotation of bacterial community was conducted using the PICRUSt algorithm within the Parallel-Meta Suite (PMS) software. This analysis uncovered various functional categories, such as metabolism, genetic information processing, and cellular processing. Overall, the findings underscore the role of the microbiome on the skin and surrounding environment in influencing the growth and health of H. kuda seahorses. Notably, this study represents the first documentation of the bacterial community inhabiting the epithelial surface of H. kuda, shedding light on its significance in the ecology of these marine organisms.

The detoxification ability of sex-role reversed seahorses determines the sexual dimorphism in immune responses to benzo[a]pyrene exposure

Sexual dimorphism in immune responses is an essential factor in environmental adaptation. However, the mechanisms involved remain obscure owing to the scarcity of data from sex-role-reversed species in stressed conditions. Benzo[a]pyrene (BaP) is one of the most pervasive and carcinogenic organic pollutants in coastal environments. In this study, we evaluated the potential effects on renal immunotoxicity of the sex-role-reversed lined seahorse (Hippocampus erectus) toward environmental concentrations BaP exposure. Our results discovered the presence of different energy-immunity trade-off strategies adopted by female and male seahorses during BaP exposure. BaP induced more severe renal damage in female seahorses in a concentration-dependent manner. BaP biotransformation and detoxification in seahorses resemble those in mammals. Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and 9-hydroxybenzo[a]pyrene (9-OH-BaP) formed DNA adducts and disrupted Ca2+ homeostasis may together attribute the renal immunotoxicity. Sexual dimorphisms in detoxification of both BPDE and 9-OH-BaP, and in regulation of Ca2+, autophagy and inflammation, mainly determined the extent of renal damage. Moreover, the mechanism of sex hormones regulated sexual dimorphism in immune responses needs to be further elucidated. Collectively, these findings contribute to the understanding of sexual dimorphism in the immunotoxicity induced by BaP exposure in seahorses, which may attribute to the dramatic decline in the biodiversity of the genus.

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.

Gene loss and co-option of toll-like receptors facilitate paternal immunological adaptation in the brood pouch of pregnant male seahorses.

Male pregnancy in syngnathids (seahorses, pipefishes, and sea dragons) is an evolutionary innovation in the animal kingdom. Paternal immune resistance to the fetus is a critical challenge, particularly in seahorses with fully enclosed brood pouches and sophisticated placentas. In this study, comparative genomic analysis revealed that all syngnathid species lost three vertebrate-conserved Toll-like receptors (TLR1, TLR2, and TLR9), of which all play essential roles in immune protection and immune tolerance in the uterus and placenta. Quantitative real-time PCR (qRT-PCR) analysis showed that the TLR paralog genes including TLR18, TLR25, and TLR21 were highly expressed in the placenta inside the seahorse brood pouch and changed dynamically during the breeding cycle, suggesting the potentially important role of the TLRs during male pregnancy. Furthermore, the immune challenge test in vitro showed a remarkable expression response from all three TLR genes to specific pathogenic antigens, confirming their immune function in seahorse brood pouches. Notably, the altered antigen recognition spectrum of these genes appeared to functionally compensate in part for the lost TLRs, in contrast to that observed in other species. Therefore, we suggest that gene loss and co-option of TLRs may be a typical evolutionary strategy for facilitating paternal immunological adaptation during male pregnancy.

Target strength of seahorse (Hippocampus kuda) based on presence dorsal and ventral aspect of brood pouch: a experiment hydroacoustic using artificial neural network

Hydroacoustic technology is a tool can be used to study the behavior of organisms. The target strength of an object is main key in hydroacoustic technology. The value of target strength is strongly influenced by level of hardness of object, in this case the seahorse has a different level of hardness between dorsal and ventral aspect. The unique of male seahorse that experiences pregnancy and keeps it’s baby in brood pouch until it hatches. This can be used as a difference between males and females based on value of target strength. Acoustic data recording using single beam scientific echosounder SIMRAD EK-15 and Sonar-4 processing software. The results showed that mean TS value of pregnant male seahorses (with brood pouch) was smaller than of female seahorses (without brood pouch), there were differences in structure composition and anatomy of the brood pouch which backscattering strength to decrease. The artificial neural network shows that there is a difference in response between seahorse (having a brood pouch/male) compared seahorse (without a brood pouch/female). The results showed that was a difference in dorsal aspect of 87.5% and ventral aspect of 98.6%. The results of this study can be used as key to distinguish sex of seahorses using the hydroacoustic method.

Characterization and expression patterns of lysozymes reveal potential immune functions during male pregnancy of seahorse

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.

Effects of pairing on color change and central gene expression in lined seahorses.

Social monogamy is a reproductive strategy characterized by pair living and defense of a common territory. Pair bonding, sometimes displayed by monogamous species, is an affective construct that includes preference for a specific partner, distress upon separation, and the ability of the partner to buffer against stress. Many seahorse species show a monogamous social structure in the wild, but their pair bond has not been well studied. We examined the gene expression of lined seahorses (Hippocampus erectus) during and after the process of pairing in the laboratory as well as color change (luminance), a potential form of social communication and behavioral synchrony between pair mates. When a seahorse of either sex was interacting with its pair mate, their changes in luminance ("brightness") were correlated and larger than when interacting with an opposite-sex stranger. At the conclusion of testing, subjects were euthanized, RNA was extracted from whole brains and analyzed via RNA sequencing. Changes in gene expression in paired males versus those that were unpaired included processes governing metabolic activity, hormones and cilia. Perhaps most interesting is the overlap in gene expression change induced by pairing in both male seahorses and male prairie voles, including components of hormone systems regulating reproduction. Because of our limited sample size, we consider our results and interpretations to be preliminary, and prompts for further exploration. Future studies will expand upon these findings and investigate the neuroendocrine and genetic basis of these behaviors.

Extensive parental care experience of male seahorses increases their future mating attractiveness.

Extensive parental care experience of male seahorses increases their future mating attractiveness.

Seahorse divorce? Of course, of course!

Long-distance, monogamous relationships are hard, regardless of how much you love your partner. Without seeing and interacting with them regularly, you may drift apart and fall for someone else. However, frequent and open communication can help keep the spark alive, even over a distance; I FaceTime my partner every night when I'm in the field. But other monogamous animals do not have cell phones or laptops; they cannot text or Zoom about their day or complain about their terrible co-workers. Without these tools, how robust are their relationships over periods of separation?To figure this out, a team of scientists from the Chinese Ministry of Agriculture and the Chinese Academy of Fishery Sciences, led by Tingting Lin, looked at lined seahorse relationships. Seahorses form monogamous pairs and mate with their partners time after time, with the males tasked with carrying around the fertilized eggs. To strengthen their bonds, paired seahorses don their finest colors and participate in synchronized swimming every day in an act known as a daily greeting, which makes my day-to-day efforts of FaceTiming my partner seem rather poor. When seahorse pairs were kept together in the lab and were able to perform their daily ritual, Lin and the team found that females usually remained faithful to their partners, even in the presence of the most attractive male seahorses, illustrating their strong bonds. However, if their male partners were injured or sick, some females sought out new partners that were healthier and better able to care for their eggs, even while their current mate was pregnant. Apparently, ‘in sickness and in health’ means nothing to seahorses.After they established that seahorse relationships tend to be strong, the team tested whether they are resilient enough to endure a break. First, Lin and the team allowed male and female seahorses to court each other, pair and mate. Then, they separated the partners for at least 12 days and introduced new males to the females, whereupon the females lost their faithfulness and mated with the new males. But it did not stop there. The scientists then separated the new male partners from the females for at least 4 days and then presented the female seahorses with the choice of either their former partner, the recent partner or a new mystery seahorse all at the same time, to see which male the females would select.But instead of preferring one of her previous partners, the female seahorse selected her new partner at random. After only a few days of separation, females showed no preference for previous partners over a random male seahorse. Even a short period of separation seems to be long enough for female seahorses to either completely forget their previous partners and the offspring they shared, or drift apart, like, well, a seahorse without seagrass to hold onto.While seahorse divorce may seem like a sad subject, it may have its benefits. If monogamous animals pair with poor quality mates, their bond, once formed, may be too strong for them to realize that their partner isn't appropriate, preventing them from seeking out better quality mates. Periods of separation may allow these animals to reassess their partners, giving them an opportunity to get out of bad relationships. So, if you do not want to end up like divorced seahorses, make sure to communicate well and/or dance in circles wearing your fanciest attire every morning.

Agonistic behaviour in the long‐snout seahorse: A gentlemanly challenge

AbstractTerritorial defence is a relatively common behaviour in reef fish. Aggression typically occurs between individuals of the same sex, given that members of the opposite sex may represent potential breeding partners. Seahorses are cryptic reef fish with typical sedentary behaviour, especially during the male pregnancy. In this context, we investigated the agonistic behaviour of pregnant seahorses, Hippocampus reidi, towards other males, pregnant or not, relating the duration of their interactions to the size and robustness of the resident and intruder. Robustness was measured as the circumference of the brood pouch divided by total body length. The study was based on four alternative treatments, which were monitored in situ: (i) a pregnant male in its home range was exposed to a pregnant intruder male; (ii) a non‐pregnant male in its home range was exposed to a pregnant intruder male; (iii) a pregnant male in its own home range was exposed to a non‐pregnant intruder male, and (iv) a non‐pregnant male in its home range was exposed to a non‐pregnant intruder male. The control to test for the effect of seahorse translocation on later displacement behaviour consisted of male seahorses translocated to an area of 60 m from the capture site and quantification of their displacement range. Low‐level aggressive behaviour was recorded in the trials, although the pregnant males presented dominance behaviour, both within and outside their own home ranges or territories, to expel intruders, while non‐pregnant males avoided interaction. No correlation was found between robustness and either the displacement of the intruder or the duration of the interaction.

High-throughput sequencing-based analysis of bacterial communities associated with Barbour's seahorses (Hippocampus barbouri) from Surigao del Norte, Philippines.

This study identified the bacterial community composition associated with Barbour's seahorses (Hippocampus barbouri). Seahorses and samples from the surrounding environment (sediment and water) were collected from Cantiasay Island in Surigao del Norte, Philippines. Genomic DNA was isolated from these samples, and the V1-V3 region of the 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. There were 929803 sequence reads corresponding to 859 operational taxonomic units (OTUs), which were obtained from the six genomic libraries. The results showed that the most abundant OTUs were affiliated to the phylum Proteobacteria, particularly those belonging to the genera Shewanella, Pseudomonas, Acinetobacter and Aeromonas, which were found to be dominant in seahorse gut samples. The only exception was for the skin of male seahorses, which was dominated by members belonging to the genus Bacillus (phylum Firmicutes). These findings on bacterial community structure and composition may shed light on therapeutic claims of Barbour's seahorses and help in the conservation of this species.

Kinematics of prey capture and histological development of related organs in juvenile seahorse

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.

Observation of male and female seahorse food types in the waters of Weh Island Indonesia

Sea horses have begun to be used as food and medicine and aquaculture efforts have begun. This study aims to observe the types of wild food consumed by each type of sea horse that lives on the waters of weh Island. The method is carried out by observation of the point VI of the station location. The results obtained were found 96 idv. Sea horses with 6 types of food (Ephausiae, Isopoda, Herpacticoida, Mysidiacea, Amphipoda and Calanoida). Both male and female sea horses The most common type of food found in the intestine is Amphipoda (43%) and the least is isopoda (0.05%).

When male seahorses take the female contraceptive pill ...

17α-ethinylestradiol (EE2), the female contraceptive pill, has been detected in mediterranean coasts where seahorse populations, Hippocampus guttulatus, live. Low environmental concentrations have the potential to disrupt growth but also endocrine metabolism, and this imbalance is all the more critical in early life stage. To investigate the impact of EE2 in reared seahorses, we exposed aged 2months and sexually undifferentiated seahorses to an environmental concentration of 21ng/L of EE2 for a period of 30days. EE2 exposure led to a 19% reduction in weight, but also a mortality rate of 27%. This exposure predicted demasculinization of male individuals with a late onset of secondary sexual characteristics. EE2 exposure led to an increase of the free androgen index, but significant reductions of estradiol and testosterone in males were observed. This low estrogen concentration seemed to impact the positive feedback on luteinizing hormone (LH) with a decrease in LH production. Added to this, synthetic estrogen had a negative impact on the production and the release of follicle-stimulating hormone. Contrary to all expectations, females demonstrated a significant decrease in vitellogenin, following exposure to EE2 at 21ng/L, while no changes were detected in males. This first study on the European long-snouted seahorses confirmed the deleterious impact of the female contraceptive pill with a real impact on growth, sexual differentiation, and maturation in young immature seahorses.

Molecular identification of GnIH and its potential role in reproductive physiology and male pregnancy of the lined seahorse (Hippocampus erectus)

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.

Sex-biased regulation of respiratory burst, phagocytic activity and plasma immune factors in lined seahorse (Hippocampus erectus) after subchronic benzo[a]pyrene exposure

Both wild and aquacultured seahorses are currently under great threat from marine pollution, notably from the potent contaminant and carcinogen benzo[a]pyrene (BaP). However, very little data are available regarding the immunomodulating effects of BaP in seahorses. Therefore, in this study, we exposed lined seahorses (Hippocampus erectus) for 7 d to BaP at three dosages (0.5, 5, and 50 μg/L) to evaluate sexual dimorphism in immune response. We measured eight immune parameters in the blood, including respiratory burst (RB), phagocytic activity (PA), monocytes/leucocytes, immunoglobulin M, complement 3, complement, interferon-a, and interleukin-2. Male seahorses had significantly higher parameters than females, except in terms of monocytes/leucocytes (P < 0.05). Although flow cytometry showed that RB and PA variation per BaP dose were roughly similar across sexes, RB and PA exhibited distinct patterns. Additionally, fluorescence intensity and leucocyte percentage were positively correlated in PA but not RB for all treatment and sex combinations. Through ELISA, we showed that the other six parameters had complex responses that nevertheless varied in a BaP-dosage and sex-dependent manner. Overall, adult male seahorses had higher immunocompetence than females before BaP exposure, and sexual dimorphism continued to be apparent during BaP exposure. Furthermore, all eight parameters were sensitive to BaP. Based on these results, we highly recommend H. erectus as a sentinel species for crude contamination, whereas PA and RB are valuable bioindicators of marine contaminants such as BaP.

The Roles of the Kisspeptin System in the Reproductive Physiology of the Lined Seahorse (Hippocampus erectus), an Ovoviviparous Fish With Male Pregnancy.

The kisspeptin/GPR54 system plays a crucial role in the regulation of the reproductive axis in vertebrates. Male pregnancy and ovoviviparity are special reproductive phenomena among vertebrates. To better understand the neuroendocrine mechanisms of male pregnancy, cDNAs encoding kiss2 and GPR54 were cloned and functionally characterized from the lined seahorse, Hippocampus erectus, an ovoviviparous teleost with male pregnancy. The core mature peptide of seahorse Kiss2 is high conserved among seahorses, but unique among vertebrate Kiss orthologs. In the phylogenic analysis, the seahorse Kiss clustered with the teleost Kiss2 clade. The kiss2 transcripts were shown to be widely expressed in various tissues, notably in the brain and gonad of the seahorse, while GPR54-2 mRNA was expressed exclusively in the brain. In addition, kiss2 mRNA found in male seahorse brain tissue increased significantly at the early pubertal stage, and decreased significantly during pregnancy. Intraperitoneal administration of seahorse Kiss2-10 to sexual mature male seahorses demonstrated to stimulate lutropin β (LHβ) and follitropin β (FSHβ) release and increased serum testosterone levels. In summary, we first identified the kisspeptin/GPR54 system in an ovoviviparous fish with male pregnancy, which might be involved in the regulation of the reproductive functions of pubertal onset, gonadal development, and male pregnancy via regulating the synthesis of both gonadotropic hormone (GTH) and testosterone.

Dimorphism of sex and gonad-development-related genes in male and female lined seahorse, Hippocampus erectus, based on transcriptome analyses

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.

Morphology of brood pouch formation in the pot-bellied seahorse Hippocampus abdominalis

BackgroundThe reproductive strategies of vertebrates are diverse. Seahorses (Pisces: Syngnathidae) possess the unique characteristic of male pregnancy; i.e., males, not females, incubate embryos in a specialized structure called a ‘brood pouch’. The brood pouch is formed along the ventral midline of the tail. The lumen of the brood pouch is surrounded by loose connective tissue, called pseudoplacenta, and dermis.ResultsWe visualized and evaluated the morphology of brood pouch formation in Hippocampus abdominalis to gain generalizable insights into this process in seahorses. First, we employed several staining methods to characterize the pseudoplacenta and dermis of the brood pouch of mature male seahorses. The pseudoplacenta is composed mainly of reticular fibers, while the dermis is composed mainly of collagenous fibers. Further observations showed that pouch formation is initiated by linear projections of epithelia on both ventrolateral sides of the body. These projections elongated toward the ventral midline, eventually fused together, and then formed a baggy structure composed of a single dermis layer with neither smooth muscle nor pseudoplacenta. Finally, the pseudoplacenta was formed, together with two layers of dermis and smooth muscle. Thus, a fully developed brood pouch was established. The morphology of the luminal epithelium also changed during pouch formation. We analyzed the localization of C-type lectins as markers; haCTL II was localized in both the outer and luminal epithelia of the brood pouch throughout development in the male seahorse, whereas haCTL IV, which was not detected in the early stage of seahorse development, became localized only in the luminal epithelium as development proceeded.ConclusionsWe categorized the processes of brood pouch formation during male seahorse development into three stages: (1) the early stage, characterized by formation of a baggy structure from the primordium; (2) the middle stage, characterized by the differentiation and establishment of brood pouch-specific tissues; and (3) the late stage, characterized by a fully formed pouch with developing blood vessels and a pouch fold ultimately capable of carrying and incubating embryos.

Plasma levels of immune factors and sex steroids in the male seahorse Hippocampus erectus during a breeding cycle.

To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11β-hydroxytestosterone (11β-OHT), 17α-methyltestosterone (17α-MT), 17β-estradiol (E2), and 17α-hydroxy-20β-dihydroprogesterone (17α-20β-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11β-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20β-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20β-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.