The electric eel (Electrophorus sp.) is an air-breathing teleost and has been known as the only fish taxon with direct vascular connections (shunts) between the ventral and dorsal aortas in all four gill arches. The aerial respiratory capillaries of the electric eel have been assumed to be perfused with the pre-branchial blood from these shunt vessels. We re-examined the cardiovascular anatomy and found these prevailing notions to be partly inaccurate. The gills of Electrophorus varii are of the holobranch structure with the afferent (ABA) and efferent (EBA) branchial arteries as discrete anatomical entities in all gill arches. Although the filaments are much shorter than in typical exclusively water-breathing teleosts, normal filamental vasculature intervenes between ABA and EBA in all arches. Yet, there are anastomoses between ABA and EBA, most notably in the fourth gill arch, where the anastomotic vessels have the same diameter as the parent vessels. There are a few, much thinner anastomotic vessels between ABA and EBA and/or the afferent and efferent filamental arteries in the first and second gill arches. The third arch has no anastomoses. The vascular space in the secondary lamellae consists of six to eight parallel interconnected vessels. Capillaries on the dorsal and lateral surfaces of the air-breathing organ are supplied by the branches of the first and second EBAs, while those on the ventral surface receive blood from the hypobranchial arteries, which originate from EBAs in all of the gill arches. Thus, the entire aerial respiratory capillaries are perfused with the post-branchial blood, not with the pre-branchial blood as previously assumed. Oxygenated blood from the air-breathing organ is drained by the anterior cardinal vein on the dorsal and lateral sides and by the inferior jugular vein on the ventral side. The heart shows no anatomical specialization to separate oxygenated and deoxygenated blood, although there is a peculiar rotation such that the bulbus arteriosus and ventricle occur on the right, while the atrium occupies the left side of the pericardial cavity. Thus, the vascular system in the head region of E. varii deviates little from the typical teleostean configuration, except for the proliferation of aerial respiratory capillaries within the air-breathing organ, a considerable degree of gill regression, and anastomoses in gill vessels. These findings, when coupled with the fact that the electric eel obtains 80% of oxygen requirement from air in normoxic water, suggest that heavy reliance on air-breathing is not a driving force for the restructuring of the central cardiovascular system toward double circulation, in which oxygenated and deoxygenated blood is separated.

Cephalopods are widely distributed in oceans worldwide and are important fishery resources. Most species have a lifespan of approximately one year and die after reproduction. In cephalopods, gonadal development may be influenced by seawater temperature; however, the endocrine mechanisms underlying reproductive maturity remain unclear. In recent years, gonadotropin-releasing hormone (GnRH)-like peptide has been identified in invertebrates, including cephalopods, as a possible endocrine regulator, similar to their role in vertebrates. Nevertheless, knowledge of its specific functions in cephalopod reproduction remains limited. This study aimed to clarify the involvement of the endogenous peptide in gonadal development in cephalopods in the bigfin reef squid (Sepioteuthis lessoniana). We performed histological observations of gonadal maturation and analyzed brain expression levels and localization of the peptide throughout sexual maturation. Additionally, we examined the relationship between annual gonadal maturation and the seawater temperature cycle. We identified coding sequences for peptides with conserved functional regions similar to those of other mollusks. Quantitative analysis revealed that brain expression significantly increased during the spermatid stage of testicular development, whereas no association with ovarian development was observed. Immunoreactivity was primarily localized in the optic lobe and around the optic gland, a central site of reproductive regulation in cephalopods. Although ovarian development progressed with increasing seawater temperature, testicular development showed no clear association with the temperature cycle. These findings suggest that GnRH-like peptides may contribute to early testicular development in S. lessoniana through optic gland signaling or direct neural pathways. In contrast, ovarian maturation appears to be strongly influenced by seawater temperature. This study provides foundational insight into the reproductive physiology of cephalopods and highlights regulatory mechanisms governing male and female gonadal development.

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we also found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may have an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on the juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may play an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

This chapter evaluates the use of zonisamide in epileptic patients with a variety of refractory seizures. It determines serum zonisamide levels, and describes the correlation between serum zonisamide levels and clinical response. Serum zonisamide levels were determined by a high-performance liquid chromatographic technique. Serum levels of other antiepileptic drugs were measured by the usual laboratory routine procedures, and the results were recorded. In epileptic children, secondary generalized epilepsy and partial epilepsy are among the most difficult to control with currently available antiepileptic drugs. Wagner et al. also observed nonlinear pharmacokinetics of this drug in adult epileptic patients after zonisamide co-medication. These aspects of both clinical response and pharmacokinetics of zonisamide resemble those of phenytoin. In conclusion, zonisamide has a broad antiepileptic spectrum. Therapeutic drug monitoring of zonisamide as well as other antiepileptic drugs seems necessary to assess clinical response and to adjust dosage.

Groupers are widely distributed in tropical and subtropical areas worldwide, are key species to coastal ecosystems, and valuable fishery targets. To facilitate artificial seed production technology for grouper aquaculture, the mechanisms of reproduction and gonad development are being elucidated for these important species. In addition, since groupers are sexually dimorphic fish with female-first maturity (protogynous hermaphrodite fish), research is being conducted to clarify the ecological mechanism of sex change and their reproductive physiology, focusing on the endocrine system. In recent years, research on groupers has also been conducted to understand changes in the coastal environment caused by ocean warming and man-made chemicals. However, due to difficulties associated with conducting research using wild populations for breeding experiments, knowledge of the physiology and ecology of these fish is lacking, especially their reproductive physiology. In this review, we present information on the reproductive physiology and endocrinology of groupers obtained to date, together with the characteristics of their life history.

To better understand the eco-physiological characteristics of reproduction in the coral reef small honeycomb grouper, Epinephelus merra, we investigated their spawning migration using biotelemetry, in addition to reproductive physiology analysis. Histological observations indicated that final ovarian maturation was not completed in honeycomb grouper collected from the coral reef pond (CRP), even during their spawning season. Additionally, our visual observations revealed that fish numbers decreased in the CRP after a full moon, which is thought to be their spawning time, suggesting their spawning migration. Next, we investigated the migration of honeycomb grouper during their spawning season using biotelemetry. Our investigations indicated that honeycomb grouper migrated from the inside of the CRP to the outside after a full moon, and then back to the inside again a few days later. These results strongly suggested that honeycomb grouper migrate to spawning sites located outside of the CRP, attain final ovarian maturation, and spawn after a full moon in the spawning season.

The blacktip grouper Epinephelus fasciatus is considered a valuable marine fish for aquaculture. This study was conducted to obtain information on its age of puberty, growth performance, gonadal development, sexual transition pattern, and plasma sex steroid levels from juvenile stage to maturity in specimens reared in captivity. The stages of gonadal development were identified by histological analysis, and plasma sex steroid levels were measured using an enzyme-linked immunosorbent assay. Our results show that maturation and spawning in blacktip groupers occurred at almost 3 years of age; although some females matured at 2 years. Plasma 17 beta- estradiol (E2) levels increased rapidly to high levels during puberty at 3 years of age, especially during the late stage of vitellogenesis at the tertiary yolk stage, and reduced at the ripe and ovulated stages prior to spawning. The gonadal somatic index also showed an increase consistent with the rise in plasma E2 levels. Plasma 11-keto-testosterone (11-KT) levels increased dramatically during sex change and in males. The significant decrease in plasma E2 levels and remarkable increase in plasma 11-KT were the key factors for the progress of sex change in these fish. Interestingly, there were the concomitant appearances of spermatocytes in ovaries of immature fe-males at 1 year 3 months of age. The earliest appearance of sex-transitioning and male fish in blackfish groupers was observed at 2 years, although no mature females were observed below this age. This indicated that the sex change and male transformation in these fish took place without them undergoing the mature female stage. In conclusion, this species had the ability to reach puberty at 2 years of age, but most individuals matured and were functional at 3 years old. Puberty might be affected by age rather than size. In addition, although this species had a protogynous hermaphrodite pattern, when individuals born in the same year were reared together, some in-dividuals were capable of directly becoming males without experiencing a mature female stage. This was due to the lack of socialization in captivity wherein individuals of various ages do not coexist, unlike that in the wild.

This study characterizes the spawning phenomena of the honeycomb grouper (Epinephelus merra), which is a lunar-synchronized spawner that spawns a few days after full moon. To elucidate the aggregation characteristics of wild honeycomb groupers, the numbers of males and females at the spawning grounds were counted before and after the full moon. Approximately 20 males were consistently observed at the spawning grounds throughout the study period. Females appeared several days after full moon and rapidly increased in number, peaking four days after full moon (41 individuals). The maturation status of the females aggregating at the spawning grounds was investigated. The gonadosomatic index increased rapidly three days after full moon, and ovulation was confirmed. Individuals with ovulatory eggs were present for three days, after which the number of females at the spawning grounds decreased. Additionally, the role of males in final oocyte maturation (FOM) and ovulation in females during the spawning phase was investigated in captivity. FOM was induced in females reared in water with mature males, suggesting that male pheromones in the water induced FOM via activation of the hypothalamic–pituitary–gonadal axis. This suggests that spawning at the natural spawning grounds was the result of male–female interactions via pheromones.

To clarify the role of estrogens in the onset of sex change in fish, estrogen levels were artificially reduced in hermaphroditic and gonochoristic female fish via the treatment with aromatase inhibitors (AIs). AI treatments caused depletion in blood estrogen levels and induced complete sex change from a female to a male in protogynous three-spotted wrasse and the honeycomb grouper. Opposite-direction sex change of the protandrous yellowtail anemonefish was also induced by AI treatments. Not only in hermaphrodites, AI treatments induced testicular differentiation and, in certain circumstances, a complete sex change in the developing ovaries of the gonochoristic fish: tilapia, medaka, zebrafish, carp and golden rabbitfish. We demonstrated that estrogen depletion induces the female to male sex change in both hermaphroditic and gonochoristic fish. Results suggested that some germ cells in the ovaries of both hermaphroditic and gonochoristic fish maintain sexual bipotentiality, which is the ability to differentiate into both female and male germ cells, throughout the life and that the fate of these germ cells' differentiation depends on the level of endogenous estrogen. Higher levels of circulating estrogen maintain femaleness, while lower levels force their differentiation into males. These studies contribute to the progress of aquaculture.

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.

Vertebrates usually exhibit gonochorism, whereby their sex is fixed throughout their lifetime. However, approximately 500 species (~ 2%) of extant teleost fishes change sex during their lifetime. Although phylogenetic and evolutionary ecological studies have recently revealed that the extant sequential hermaphroditism in teleost fish is derived from gonochorism, the evolution of this transsexual ability remains unclear. We revealed in a previous study that the tunica of the ovaries of several protogynous hermaphrodite groupers contain functional androgen-producing cells, which were previously unknown structures in the ovaries of gonochoristic fishes. Additionally, we demonstrated that these androgen-producing cells play critical roles in initiating female-to-male sex change in several grouper species. In the present study, we widened the investigation to include 7 genera and 18 species of groupers and revealed that representatives from most major clades of extant groupers commonly contain these androgen-producing cells, termed testicular-inducing steroidogenic (TIS) cells. Our findings suggest that groupers acquired TIS cells in the tunica of the gonads for successful sex change during their evolution. Thus, TIS cells trigger the evolution of sex change in groupers.

The subtypes of zona pellucida (zp), primarily expressed in female gonads, are considered novel molecular markers for testis-ova (or intersex), a type of gonadal abnormality caused by environmental estrogens (EEs) in Japanese medaka (Oryzias latipes). However, the association between testis-ova and the expression of gonadal zp subtypes is unclear in other teleost species, particularly in species studied in field surveys. In this study, 17 alpha ethinylestradiol (EE2) was orally administrated at 4-4000 ng/g body weight (BW)/day for 28 days to gray mullets (Mugil cephalus), and gonadal abnormalities were studied using histological analysis. The expression profiles of gonadal zp subtypes (zpb and zpc5) were analyzed to evaluate their suitability as gonadal abnormality markers by comparing with a hepatic vitellogenin (vtg) subtype (vtgAb). The oral administration of EE2 40 and 400 ng/g BW/day for 28 days induced significant gonadal zpb expression, and the gonads showed moderate abnormality (testis-ova). Conversely, the gonadal zpc5 levels decreased significantly in response to the oral administration of EE2 at 4000 ng/g BW/day for 28 days, and the gonads exhibited severe abnormalities. The hepatic vtgAb levels increased upon EE2 treatment regardless of gonadal abnormality. Therefore, the gonadal zpb levels and hepatic vtgAb levels served as appropriate markers for testis-ova and EE2 presence, respectively. However, the diagnosis of severe gonadal abnormality using gonadal zpc5 was moderately accurate. The findings suggest that the combination of vtgAb, zpb, and zpc5 is a potential marker for gonadal abnormality caused by EE contamination in gray mullet. That said, the potential of zpc5 should be reconsidered to determine if it shows greater accuracy in a larger or more diverse population.

Gonadotropin-releasing hormone (GnRH) is one of the most important neuroendocrine regulators for animal reproduction. GnRH-like peptide (GnRH-like) has recently been shown to play a critical reproductive role mainly in gametogenesis or steroidogenesis in the gonads of some molluscs, including cephalopods. However, its involvement in gonadal sex differentiation remains unknown. Here, we show the expression profile of GnRH-like in the brain of the cephalopod kisslip cuttlefish, Sepia lycidas, throughout gonadal sex differentiation, by quantitative real time RT-PCR and immunohistochemistry. We found that GnRH-like could be detected in the brain at a sexually undifferentiated stage, and its expression level significantly increased upon initiation of gonadal sex differentiation. However, no significant difference in GnRH-like expression levels was observed between sexes during gonadal sex differentiation. Additionally, we demonstrated immunoreactivity of GnRH-like in glial cells or immature neurons, which are mainly distributed in the non-reproductive related area of the cephalopod brain, suggesting the immature function of the reproductive endocrine axis during early ontogenesis. Our results demonstrate for the first time, the expression profile of GnRH-like during early ontogenesis in cephalopods.

Groupers in subfamily Epinephelinae, family Serranidae, are highly valuable marine resources, especially in Southeast Asia including Japan. The objective of this study was to understand the morphological characteristics and endocrine mechanisms underlying the sex diŠerentiation and sex change in groupers. First, we analyzed the morphological characteristics and the role of sex steroid hormones on the gonadal sex diŠerentiation using the large Malabar grouper, Epinephelus malabaricus. Next, the physiological mechanism of sex change was revealed by using the small Honeycomb grouper, E. merra, as a model ˆsh. Finally, we succeeded in inducing an artiˆcial sex reversal from female to male in the groupers by using aromatase inhibitor or gonadotropin; this is in addition to the alreaady-existing methods that use androgen and could be achieved by applying the basic information about the physiological mechanism of sex change in groupers.

To understand and obtain basic information on sex differentiation in the kisslip cuttlefish (Sepia lycidas), the gonadal sex differentiation process was investigated histologically. An undifferentiated gonad consisting of germ cells and somatic cells was found to form at a caudal site in the space between the internal yolk sacks of cuttlefish embryos at 14 and 21 days after spawning (DAS). Sexual dimorphism in the gonad was first detected at around 28 DAS. Meiotic oocytes were observed as the first visible morphological characteristic of ovaries in the gonads of some cuttlefish embryos at 28 DAS. In other individuals, neither meiotic germ cells, nor the appearance of a testicular structure, were observed in the gonad even after 10 days post hatching (DPH). Seminiferous tubules, consisting of a small number of spermatogonia and a surrounding basement membrane, were the first visible morphological characteristic of the testis in the male gonad, detected at around 20 DPH. This is the third report on the gonadal sex differentiation process in cephalopods.

To clarify the cause of sex change recovery after the withdrawal of androgen treatment, immature female Malabar grouper were fed a diet containing 17alpha-methyltestosterone (MT) at 50 μg/g for 7 mo and then a normal diet for 6 mo. The MT brought about precocious sex change from immature ovaries to mature testes with active spermatogenesis, including the development of spermatozoa, and sex change reversed soon after MT treatment withdrawal. This result indicates that precocious sex change in immature Malabar grouper with oral MT treatment is impermanent. The expression of three steroidogenic enzymes (Cyp11a, Cyp19a1a, and Cyp11b) in the gonads of the Malabar grouper were analyzed immunohistochemically at the end of the 7-mo treatment. No apparent differences were seen in the expression pattern of these enzymes between the mature testes of MT-treated fish and the immature ovaries of control fish. In addition, serum estradiol-17beta and 11-ketotestosterone levels in treated fish were the same as those in control fish. These results indicate that in the case of immature Malabar grouper MT might have little effect on endogenous steroidogenesis during precocious sex change even though it induced active spermatogenesis in the gonads of treated fish. From these results, we also concluded that MT might have little effect on the steroidogenic endocrine pathway, and this is one cause of sex change recovery after treatment withdrawal.

Grouper culture assumes importance like any other economically important food fish aquaculture practice in Japan. Regular availability of sexually mature brood stock at appropriate time and stable seed production are considered vital for prolific fish farming. Despite the fact that previous studies have induced sexual maturation and spawning in some grouper species that had ovaries with vitellogenic or full-grown mature oocytes, little attention has been paid to artificial induction of sexual maturation and spawning of immature fish in off-spawning season. Developing such a method will promote grouper aquaculture immensely. In an aim to facilitate such a task, we used one of the popular experimental model fishes, the honeycomb grouper, Epinephelus merra, to induce sexual maturation during non-breeding season by modulating environmental factors with gonadotropin-releasing hormone analogue (GnRHa) implantation using osmotic pump. We performed three different experiments flanking various periods of non-breeding season under artificial/natural photothermal conditions with or without GnRHa implantation. Induction of sexual maturation is scored by the presence of vitellogenic to mature oocytes after various treatments in addition to the data of gonado-somatic index and plasma estradiol levels. Among various treatments, high water temperature with long photoperiod conditions induced sexual maturation in immature groupers during October to December. Implantation of GnRHa to these fishes induced sexual maturation at all periods of non-breeding season tested in this study. In fact some of the fishes belonging to these groups have even spawned during the course of treatment indicating the effectiveness of the treatments. On the other hand, neither GnRHa implantation alone nor exposure to natural photothermal conditions evoked sexual maturation. Likewise, combination of short and long photoperiod with low to high water temperature exposure over the duration period was found to be least effective with or without GnRHa implantation. Similarly, exposure to long photoperiod or high water temperature alone did not induce sexual maturation. Hence, combination of artificial photothermal conditions with GnRHa implantation seems to be a decisive method to induce sexual maturation in immature fish. Based on these results, we demonstrate a superior strategy for successful breeding of sexually immature marine teleost fish during non-breeding season by modulating environmental variables with GnRHa implantation.

The aim of this study was to clarify the roles of 2 gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on sex differentiation in the protogynous Malabar grouper, Epinephelus malabaricus. To do this, the mRNA expression patterns of GTH subunits (cga, fshb, and lhb) in the fish pituitary throughout gonadal sex differentiation were investigated. Real-time reverse transcriptase (RT)-PCR showed that cga and fshb were present in the undifferentiated and ovarian differentiation stages, and that the expression levels significantly increased after ovarian differentiation (AOD). However, lhb was not expressed before ovarian differentiation (BOD) and was first detected AOD. Next, to investigate the differentiation and distribution of Fshb and Lhb-producing cells in the pituitary of fish throughout gonadal sex differentiation, immunohistochemical analysis was used to detect teleost GTH subunits. Positive immunoreactivity against Fshb and Lhb was not detected in the pituitary BOD; Fshb and Lhb-positive cells first appeared in the pituitary AOD. It therefore seems unlikely that pituitary gonadotropins play a major role in the control of gonadal sex differentiation in the Malabar grouper.