Neuroanatomy, Na⁺/K⁺-ATPase distribution, and melanomacrophage centers in the brain of pond-reared female doublespotted queenfish, Scomberoides lysan (Perciformes: Carangidae)

ABSTRACT

Fish neurobiology, neuromorphology, and histology are crucial to the development of aquaculture. However, the neurobiology of the doublespotted queenfish, Scomberoides lysan (Forsskål, 1775), a candidate species for aquaculture in Thailand, has not been investigated. Five captive S. lysan females were examined to characterize brain morphology, Na⁺/K⁺-ATPase (NKA) activity, and the presence of melanomacrophage centers (MMCs). Brains were collected for morphological and histological observations and immunohistochemical assays. A large optic tectum and a moderately sized cerebellum were identified among the brain regions. The optic tectum comprised six layers: the stratum marginale, stratum opticum, stratum fibroetgriciale, stratum album centrale, stratum griseum centrale, and stratum periventriculare. The cerebellum comprised three layers: the outer molecular layer, Purkinje cell layer, and inner granular layer. Purkinje cell density did not differ significantly between the corpus cerebelli (6.8 ± 0.66 per area) and valvula cerebelli (6.3 ± 0.89 per area). MMCs were scattered in connective tissue associated with blood vessels and in proximity to the nucleus lateralis tuberis. NKA immunoreactivity was detected in the plasma membranes of neurons and neuronal fibers across several brain regions. This study provides the first histomorphological characterization of the S. lysan brain and offers neurobiological insights relevant to reproductive physiology (brain-pituitary-gonad axis) and broodstock management in captivity.

KEYWORDS
Aquaculture; central nervous system; histology; immunohistochemistry; Thailand

INTRODUCTION

Physiological microstructures in the vertebrate brain have been well studied. Purkinje cells in the cerebellum play a prominent role in motor coordination, balance, and various aspects of cognition (Calvo and Schluessel 2021, Kotrschal et al. 1998, Pollen et al. 2007). In fish, the brain varies in size and structure (Wagner 2003), and brain morphology has long been linked with behavior, habits, and ecological contexts (Kotrschal et al. 1998, Kharlamova and Saveliev 2019, El-Ghazali et al. 2023). Brain size and structure have been widely reported in Rastrelliger brachysoma (Bleeker, 1851) (Senarat et al. 2016), Mugil cephalus Linnaeus, 1758 (Hussein and Cao 2019), Hippocampus barbouri Jordan & Richardson, 1908 (Senarat et al. 2020), and Clarias gariepinus (Burchell, 1822) (El-Ghazali et al. 2023), with all published data demonstrating the adaptive significance of brain organization for behavior.

Brain structures play an important role in supporting gonadotropin-releasing hormone (GnRH) neuron systems and the functions of hypophysiotropic GnRH neurons in the fish brain (Karigo and Oka 2013, Christian and Moenter 2010). Several fish species, such as Coregonus clupeaformis (Mitchill, 1818) (Adams et al. 2002) and Astatotilapia burtoni (Günther, 1894) (Greenwood and Fernald 2004), have shown that GnRH activity is responsible for steroid synthesis pathways (Honji et al. 2015) and reproductive performance (Honji et al. 2019). Brain biology should therefore be investigated to resolve reproductive dysfunction in captive fishes, particularly under ongoing technological improvements for aquaculture (Zohar and Mylonas 2001, Zohar et al. 2010, Mitparian et al. 2023).

Melanomacrophage centers (MMCs) are a distinctive group of pigment-containing phagocytic cells in vertebrates (Agius and Roberts 2003). MMCs have been found in the hematopoietic organs and liver of numerous teleosts as part of the physiological immune system (Agius and Roberts 2003, Steinel and Bolnick 2017). Rønneseth et al. (2015) used histology and transcriptomics to demonstrate that MMCs play a key role in the immunological response of fish. MMCs are also used as biomarkers of fish welfare and health in environmental monitoring programs related to pollution (Agius and Roberts 2003) and stressor conditions (Passantino et al. 2002, 2024). Increased MMC density in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), has been associated with exposure to a diverse range of stressors (Passantino et al. 2002, 2004), and increased MMC density in the liver parenchyma of Silurus herzbergii (Bloch, 1794) from São José Bay was associated with reduced fish health (Viana et al. 2021).

Sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase or NKA) is a ubiquitous membrane-bound protein complex consisting of a catalytic α-subunit with a molecular weight of 100 kDa, a glycosylated β-subunit of 55 kDa, and a smaller γ-subunit with a tissue-specific FXYD protein (Geering 2005). NKA modulation is a crucial component of Na⁺ and K⁺ transport across the plasma membrane, regulating ionic gradients and homeostasis (Geering 2005). NKA activity in fishes has been investigated in various tissues, including the NKA α-subunit in the kidneys of Danio rerio (Hamilton, 1822) (Esbaugh et al. 2019) and Tetraodon nigroviridis nigroviridis (Lafresnaye, 1843) (Lin et al. 2004) and throughout the gill epithelium (Hwang and Lee 2007, Wang et al. 2011); however, research into NKA activity in the fish brain has been limited (Peter and Simi 2017, Otero-Rodino et al. 2019). NKA activity in the brain of O. mykiss has been associated with the regulation of food intake (Otero-Rodino et al. 2019).

The doublespotted queenfish, Scomberoides lysan (Forsskål, 1775) (Perciformes: Carangidae), is an economically important marine fish found in estuaries and coastal waters across the Indo-Pacific region (Thulasitha and Sivashanthini 2012). The descriptive morphology of the species has been investigated (Kimura et al. 1998), and its reproductive biology has been recorded (Thulasitha and Sivashanthini 2013); however, despite the functional and developmental importance of the nervous system in fish, the nervous system of S. lysan has never been reported. In this study, only female S. lysan were used, given the role of female neurobiology in regulating egg quality, fecundity, and overall farm profitability through hormonal pathways. Here, the foundational features of the S. lysan brain, together with evidence of NKA activity and MMC presence, were investigated using morphological and histological methods and immunohistochemistry. These results provide the first neurobiological insights into the basic neurobiology of this species and establish a foundation for understanding the links between brain physiology, GnRH neuron systems, and reproductive performance in S. lysan, with direct implications for aquaculture development.

MATERIAL AND METHODS

Five outwardly healthy S. lysan individuals with a mean total length of 34.0 ± 0.9 cm were selected from the rearing ponds of the Faculty of Science, Rajamangala University of Technology Srivijaya, Trang Campus, in September 2020. One hundred wild juvenile S. lysan of approximately 7.5 cm total length were purchased from local fishermen along the Andaman Sea coastline in Thailand and reared for 18 months prior to further observations. The stocking density was approximately 100 individuals per pond, providing an average area of 4.2 m² per fish. The fish were fed freshly chopped yellowtail scad, Selaroides leptolepis (Cuvier, 1833) instead of commercial pellets and were hand-fed twice daily, at 8:00 a.m. and 4:00 p.m., at a feeding rate of 8% of body weight per day. All S. lysan were maintained under controlled conditions, with salinity ranging from 25 to 30 ppt, water temperature from 28 to 30 °C, and dissolved oxygen remaining above 3 mg/L. Taxonomic identification followed FAO (1983).

Resected brain samples were examined morphologically under an SZX12 stereomicroscope and photographed with an Olympus DP11 digital camera. Scientific illustrations were created with Adobe Illustrator CS5. Brain length and width were measured for each sample and expressed as mean ± SEM.

Fresh brain samples were fixed in Davidson’s fixative for 48 hours at room temperature, and five cross-sections of each brain (from telencephalon to myelencephalon) were collected. All sections were processed following a standard paraffin histological protocol (Presnell and Schreibman 2013, Suvarna et al. 2019). Three sections of 4 µm thickness were taken from each cross-section, mounted on microscope slides, and stained with Harris’s hematoxylin and eosin (H&E). Histological slides were examined under light microscopy and photographed using a 3DHISTECH Panoramic Viewer (3DHISTECH, Hungary).

Ten random digital images at 40× magnification from each stained histological slide were selected, and capillary density was calculated in the cerebral hemisphere, optic tectum, thalamus, cerebellum, and medulla oblongata using ImageJ software. The number of Purkinje cells per image was also counted. All data are reported as means ± SEM and were analyzed and plotted with Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Data normality was assessed using the Shapiro-Wilk test. Capillary density among brain regions was compared by one-way ANOVA followed by Tukey’s post hoc test for normally distributed data and Dunn’s post hoc test when data were not normally distributed. Purkinje cell density between cerebellar regions was compared using Student’s t-test. Differences were considered statistically significant at p < 0.05.

The primary anti-NKA α1 antibody (a5-s, Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA, USA) was used to detect NKA. Unstained brain sections were deparaffinized, rehydrated, and submerged in 0.5% Triton X-100/PBS for 30 min, then blocked with 0.3% H₂O₂ for 10 min to quench endogenous peroxidase activity. Sections were incubated at room temperature for two hours with Blocking One solution (03953-95, Nacalai Tesque, Kyoto, Japan) to prevent nonspecific binding. After blocking, sections were immunoreacted overnight at 4 °C with the anti-NKA α1 antibody at a 1:5 dilution in Blocking One solution, following the protocol of Senarat et al. (2025). The following day, sections were incubated for two hours at 4 °C with horse anti-mouse IgG antibody (H+L) conjugated to horseradish peroxidase (PI-2000, Vector Laboratories, Burlingame, CA, USA) at a 1:500 dilution in 0.1% Tween-20/PBS. Sections were then incubated for five minutes at room temperature with 3,3’-diaminobenzidine tetrahydrochloride (DAB; ImmPACT DAB Peroxidase Substrate Kit SK-4105, Vector Laboratories) as the chromogenic substrate. Negative controls were prepared by omitting the primary antibody. Brain sections showing NKA immunoreactivity were photographed using the same equipment described above.

RESULTS

Morphology and histological organization of the brain

The brain was located in the cranial cavity and comprised five regions: the telencephalon, mesencephalon, diencephalon, metencephalon, and myelencephalon. A dorsal view showed that the two telencephalic hemispheres were the smallest portion of the brain (approximately 0.58 ± 0.02 cm in width and 0.58 ± 0.03 cm in length; Fig. 1A-C). The optic tectum in the mesencephalon comprised two distinct bilateral lobes, forming the largest visual area (approximately 0.96 ± 0.02 cm in length) (Fig. 1A-C). A moderately sized cerebellum (approximately 0.65 ± 0.08 mm) was identified in the metencephalon (Fig. 1A-C).

Figure 1
Gross morphology and morphometric measurements (width and length) of the Scomberoides lysan brain. (A-C) Dorsal view showing the cerebral hemisphere (Cb), optic tectum (Ot), cerebellum with corpus cerebelli (Cc) and valvula cerebelli (Vc), vagal lobe (Vl), and medulla oblongata (Md). The histogram in (B) shows mean dimensions (± SE) of the telencephalon, optic tectum, and cerebellum. (D-G) Diagram and stained cross-sections of the brain showing the cerebral hemisphere (Cb), optic tectum (Ot), diencephalon (Di) with epithalamus (Epi), thalamus (Ta), and hypothalamus (Hy), pituitary gland (Pg), and cerebellum with corpus (Cc) and valvula (Vc) cerebelli. Ar = anterior; Pr = posterior. Staining: Harris’s hematoxylin and eosin (H&E). Scale bars: A, G = 1 mm; E, F = 500 µm.

Cross-sections from anterior to posterior revealed several distinct brain regions, including the cerebral hemis­phere, optic tectum, three regions of the diencephalon (epitha­lamus, thalamus, and hypothalamus) together with the pituitary gland, two regions of the cerebellum (corpus and valvula cerebelli) (Figs 1D-2F), and the medulla oblongata associated with the cranial nerve (Fig. 2G-H).

Figure 2
Cross-sections of the Scomberoides lysan brain at the light microscopic level. (A-H) Serial cross-sections from anterior to posterior showing the cerebral hemisphere (Cb), optic tectum (Ot), corpus cerebelli (Cc), valvula cerebelli (Vc), vagal lobe (Vl), medulla oblongata (Md), and cranial nerve (Cn). Ar = anterior; Pr = posterior. Staining: H&E. Scale bars: B = 1 mm; D, C = 500 µm; E, G, H = 200 µm; F = 100 µm.

Brain histology and Na⁺/K⁺-ATPase distribution

The telencephalon contained diverse neuroglia and blood vessels (Fig. 3A-C) and oval neurons with a central nucleus (Fig. 3D). No NKA immunoreactivity was detected in the cells or blood vessels of this region (Fig. 3E-F; nega­tive control Fig. 3G). The optic tectum displayed a highly developed multilayered structure (Fig. 4A) similarly organized in both lobes (Fig. 4B-C). Neuronal fibers in the optic tectum reacted positively to the NKA antibody (Fig. 4D; negative control in Fig. 4E). The diencephalon comprised three distinct zones arranged from dorsal to ventral: the epithalamus, thalamus, and hypothalamus (Fig. 4F-G). Neurons in the dorsal epithalamus (Fig. 4H) expressed NKA (Fig. 4I), as did neurons in the habenular ganglion (Figs 4J-K), which reacted positively with the NKA antibody (Fig. 4L; negative control (Fig. 4M). The caudalmost part of the inferior lobes of the hypothalamus harbored the saccus vasculosus, a specialized circumventricular organ (Fig. 4N), lined with pseudostratified cuboidal epithelium (Fig. 4O). MMCs and red blood vessels were also evident in this region (Fig. 4O).

Figure 3
Cross-sections of the cerebral hemisphere of Scomberoides lysan at the light microscopic level. (A) The two lobes of the cerebral hemisphere. (B-C) Neuroglia (Ng), neurons (Nu), and blood vessels (Bv). (D) High magnification showing neuronal structure with central nucleus (N). (E-F) Absence of NKA immunoreactivity in cells and blood vessels of the telencephalon. (G) Negative control. Staining: (A-D) H&E; (E-G) NKA immunohistochemistry. Scale bars: A = 200 µm; B, E, G = 10 µm; C = 20 µm; D, F = 5 µm.

Figure 4
Cross-sections of the optic tectum (Ot) and diencephalon (Di) of Scomberoides lysan at the light microscopic level. (A) The optic tectum positioned above the corpus cerebelli (Cc). (B-C) Higher magnifications of the boxed areas showing distinct layers of the optic tectum: 1. meninx; 2. stratum marginale; 3. stratum opticum; 4. stratum fibroetgriciale; 5. stratum album centrale; 6. stratum griseum centrale; 7. stratum periventriculare. (D) NKA immunoreactivity in neuronal fibers (arrows). (E) Negative control. (F-G) The diencephalon comprising epithalamus (Epi), thalamus (Ta), and hypothalamus (Hy). (H-I) Neurons (Nu) in the dorsal epithalamus showing NKA expression. (J-K) Habenular ganglion (Ha) with neurons (Nu) and neuroglia. (L) Positive NKA reaction at neuronal cell membranes (asterisks). (M) Negative control. (N-O) Saccus vasculosus (Sv) lined with pseudostratified cuboidal epithelium (Pce) and blood vessels (Bv). Staining: (A-C, F-H, J-K, N-O) H&E; (D-E, I, L-M) NKA immunohistochemistry. Scale bars: A, G = 500 µm; B, C, J = 100 µm; D, E = 50 µm; F = 1 mm; H, N = 20 µm; I, L, M = 10 µm; K, O = 5 µm.

Several nuclei in the hypothalamus (Fig. 5A), including the nucleus preopticus (NP; Fig. 5B), contained neurons (Fig. 5C) that reacted positively with the NKA antibody (Fig. 5D; negative control (Fig. 5E). Neurons and neuroglia were also identified in the nucleus periventricularis (NPV; Fig. 5F) and the nucleus lateralis tuberis (NLT; Fig. 5G). The pituitary gland was histologically continuous with the hypothalamus (Fig. 5H) and comprised the adenohypophysis and neurohypophysis (Fig. 5H-I). Three cell types were identified in the adenohypophysis: acidophilic cells, basophilic cells, and chromophobe cells (Fig. 5J).

Figure 5
Cross-sections of the diencephalon (Di) and pituitary gland of Scomberoides lysan at the light microscopic level. (A) Hypothalamic nuclei: nucleus preopticus (NP), nucleus lateralis tuberis (NLT), and nucleus periventricularis (NPV). (B) Neurons in the NP. (C) Neurons in the NPV. (D) NKA immunoreactivity in neurons (Nu). (E) Negative control. (F-G) Neurons and neuroglia in the NLT. (H-I) Pituitary gland (Pg) comprising the adenohypophysis (Ad) and neurohypophysis (Neu; dashed lines). (J) High magnification showing acidophilic cells (red dashed line), basophilic cells (long arrow), and chromophobe cells (short arrow) in the adenohypophysis. Hy = hypothalamus. Staining: (B-C, F, H-J) H&E; (D-E) NKA immunohistochemistry. Scale bars: B, D, J = 10 µm; C, E = 5 µm; F = 20 µm; H = 100 µm; I = 50 µm.

The cerebellum, the main component of the metencephalon, was positioned beneath the optic tectum (Fig. 6A) and comprised the corpus and valvula cerebelli (Fig. 6A). All cerebellar regions shared a tightly folded layered organization, except at the junction with the medulla oblongata (Fig. 6A). Three major neuronal layers were identified: the outer molecular layer, the Purkinje cell layer, and the inner granular layer (Fig. 6B-C). At high magnification, Purkinje cells appeared large and pear-shaped, with elongated axons (Fig. 6D). Purkinje cell density was 6.8 ± 0.66 per area in the corpus cerebelli (n = 10 areas) and 6.3 ± 0.89 per area in the valvula cerebelli (n = 10 areas) (Fig. 6E), with no significant difference between regions. Purkinje cell cytoplasm and cell membranes reacted positively with the NKA antibody (Fig. 6F-H; negative control in Fig. 6I).

Figure 6
Cross-sections of the cerebellum and morphometric diagram of Purkinje cell density in Scomberoides lysan. (A) The cerebellum comprising corpus cerebelli (Cc) and valvula cerebelli (Vc), positioned above the medulla oblongata (Md). (B-C) High magnifications of the Cc and Vc showing three layers: granular layer (Gl), Purkinje cell layer (Pc), and molecular layer (Ml). (D) Large pear-shaped Purkinje cells (arrows) with basophilic cytoplasm. (E) Histogram showing Purkinje cell density (means ± SE) in the Cc and Vc; no significant difference was observed between regions (p > 0.05). (F-G) NKA immunoreactivity in Purkinje cell cytoplasm (asterisks) and cell membranes (double asterisks). (I) Negative control. Staining: (A-D) H&E; (F-G, I) NKA immunohistochemistry. Scale bars: A = 500 µm; B, F, I = 10 µm; C, D, G = 5 µm.

The medulla oblongata, part of the myelencephalon, contained clusters of neurons associated with axonal processes (Fig. 7A-B). These neurons reacted positively with the NKA antibody (Fig. 7C; negative control in Fig. 7D). Cranial nerve fibers were also identified (Fig. 7E-F) and expressed NKA immunoreactivity (Fig. 7G; negative control in Fig. 7H).

Figure 7
Cross-sections of the medulla oblongata (Md) and cranial nerve (Cn) of Scomberoides lysan at the light microscopic level. (A-B) Large neurons (Nu) and axons (Ax) in the medulla oblongata. (C) NKA immunoreactivity in neurons (Nu). (D) Negative control. (E) Cranial nerve (Cn) associated with the medulla oblongata. (F-G) High magnification showing neuronal fibers (arrows) and their NKA immunoreactivity (asterisks). (H) Negative control. Staining: (A-B, E-F) H&E; (C-D, G-H) NKA immunohistochemistry. Scale bars: A, C, D, F = 20 µm; B = 10 µm; E = 200 µm, G, H = 5 µm.

Morphometric analysis of capillaries

Capillaries were identified in the cerebral hemispheres, optic tectum, hypothalamus (Fig. 8A), cerebellum, and medulla oblongata (Fig. 8B-C). Capillary density was compared among these brain regions (Fig. 8D). Capillary density was highest in the hypothalamus, but did not differ significantly among regions (Fig. 8D).

Figure 8
Capillary distribution and melanomacrophage centers (MMCs) in the Scomberoides lysan brain at the light microscopic level. (A-C) Capillaries (Cp) in close proximity to neuroglia (arrows) and neurons (Nu). (D) Histogram showing capillary density (means ± SE) in different brain regions; no significant differences were observed among regions (p > 0.05). (E-F) MMCs in the hypothalamus and saccus vasculosus, respectively. (G) Scattered MMCs in connective tissue (CNT) adjacent to blood vessels (BV). (H) Scattered MMCs in other areas of the brain parenchyma. White arrows indicate erythrocytes. Staining: (A-C, E-H) H&E. Scale bars: A, C, E = 5 µm; B = 100 µm; F, G, H = 20 µm.

Melanomacrophage centers

Melanomacrophage centers were observed in all brain samples, mainly in connective tissue adjacent to blood vessels, the nucleus lateralis tuberis (Fig. 8E-F), and the saccus vasculosus (Fig. 8G). Scattered MMCs were also observed in other areas of the brain parenchyma (Fig. 8H).

DISCUSSION

This study provides the first comprehensive neuroanatomical and histological characterization of the brain of female S. lysan, together with an initial investigation into NKA distribution and MMC presence. These findings offer baseline data enabling insights into the neuroecology of the species, potential indicators for health assessment, and directions for future aquaculture development and management.

Neuroanatomical correlations of neuroecology and behavior

The gross morphology of the S. lysan brain is characterized by a prominently large optic tectum and a moderately sized cerebellum. This finding aligns with observations in other visually oriented, active pelagic or coastal marine teleosts, such as R. brachysoma (Senarat et al. 2016), M. cephalus (Hussein and Cao 2019), H. barbouri (Senarat et al. 2020), and C. gariepinus (El-Ghazali et al. 2023). The optic tectum, comprising six distinct well-organized layers, underscores the importance of vision for this species. Similar multilayered optic tectum organization has been reported in Rineloricaria heteroptera Isbrücker & Nijssen, 1976 (Angulo et al. 2017), M. cephalus (Hussein and Cao 2019), H. barbouri (Senarat et al. 2020), and Paracheirodon axelrodi (Rincón et al. 2017). This intricate structure is essential for processing complex visual information to support open-water behaviors such as schooling, prey detection and capture, and predator avoidance (Pollen et al. 2007, Park and Bell 2010, Corral-Lopez et al. 2023,).

The moderately sized cerebellum, with its characteristic three-layered structure and NKA-positive Purkinje cells, suggests a well-developed capacity for motor coordination, balance, and motor learning (Rodriguez et al. 2005, Chang et al. 2020). Cerebellar volume has also been correlated with cognitive flexibility and the execution of complex motor sequences linked to problem-solving (Rodriguez et al. 2005, Triki et al. 2022). High magnification of cerebellar histology revealed large pear-shaped Purkinje cells with elongated axons (Fig. 6D). This morphology has been well characte­rized in mormyrid fish (Russell and Bell 1978, Zhang et al. 2011) and zebrafish (Chang et al. 2020). Recent evidence from zebrafish has associated the cerebellum with both locomotor and non-locomotor functions (Chang et al. 2020, 2021), with Purkinje cells in the corpus cerebelli responding specifically to temporal activity during locomotion (Chang et al. 2020). The corpus cerebelli of S. lysan may therefore be a major site controlling locomotor functions involved in swimming behavior.

While basal survival behaviors are controlled by lower brain structures such as the brainstem, the forebrain and cerebellum translate these reflexes into adaptive learned responses, including nuanced threat assessment and flexible avoidance tactics (Portavella et al. 2004, Triki et al. 2022). The combination of a dominant optic tectum and a functional cerebellum suggests that S. lysan is well-adapted for translating complex visual input into rapid, precise movements required for hunting mobile prey and navigating dynamic social environments such as schooling. This neuroanatomical organization strongly supports its known ecological role as an active piscivore in coastal and estuarine waters (Qamar et al. 2018), likely relying heavily on visual cues for prey detection, predator avoidance, and social interactions (Yoshida et al. 2013).

Hypothalamic structures: integrating environment, reproduction, and aquaculture

The saccus vasculosus, a specialized circumventricular organ, was located in the caudalmost region of the hypothalamic inferior lobes. This finding is consistent with previous reports in Scyliorhinus canicula (Turkmen et al. 2007) and Epinephelus coioides (Sharareh et al. 2013). The saccus vasculosus was lined with pseudostratified cuboidal epithelium, and both MMCs and red blood vessels were evident in this region. The function of this organ remains incompletely understood; it has been proposed to act as a sensor of seasonal changes in photoperiod (Nakane et al. 2013) and to be involved in neuropeptide release (Sueiro et al. 2007).

Histological examination also revealed that the NLT and NPV contain prominent neuronal and neuroglial populations with extensive capillary networks. These structural features are consistent with their roles in gonadotropin-releasing hormone (GnRH) secretion and reproductive regulation. The NLT contains dopaminergic neurons that exert inhibitory control over GnRH1 and GnRH3 release under stress or suboptimal conditions, providing the neuroanatomical basis for stress-induced reproductive suppression in fishes (Li et al. 2022, Sun et al. 2022, Di Yorio et al. 2019). The NPV produces gonadotropin-inhibitory hormone (GnIH), which suppresses GnRH3 neurons and reduces pituitary gonadotropin sensitivity (Muñoz-Cueto et al. 2017, Di Yorio et al. 2019). Through these dual projections, the NPV coordinates inhibitory signals across multiple levels of the reproductive axis (Paullada-Salmerón et al. 2019, Di Yorio et al. 2019).

The specialized architecture of both nuclei enables the integration of environmental signals, including photoperiod and stress, into coordinated reproductive and growth responses (Li et al. 2022, Di Yorio et al. 2019). NLT dopaminergic activity increases during non-breeding seasons to suppress gamete development (Robinson et al. 2009), and chronic stress can enhance this inhibitory tone through NLT neuron hypertrophy and increased inhibitory hormone signaling (Ganesh 2021, Honji et al. 2013, Di Yorio et al. 2019). NPV-driven GnIH expression is regulated by photoperiod via melatonin-sensitive pathways, with longer days reducing inhibition to permit spawning in teleost fishes (Nakane et al. 2013). These nuclei also mediate a trade-off with growth, as GnIH can either stimulate or inhibit growth hormone (GH) release depending on the species - stimulating GH in sockeye salmon, Oncorhynchus nerka (Amano et al. 2006), and Nile tilapia, Oreochromis niloticus (Ogawa et al. 2016), but inhibiting it in European sea bass, Dicentrarchus labrax (Paullada-Salmerón et al. 2016).

This inhibitory system represents a major bottleneck in captive breeding programs. Stress from rearing conditions such as confinement and handling can chronically activate these inhibitory pathways, leading to reproductive dysfunction (Sun et al. 2022). Understanding the structural and functional relationships within the NLT and NPV has significant practical implications for aquaculture. GnRH analogue (GnRHa) therapies have been developed to overcome this bottleneck by providing a potent artificial stimulus to the pituitary (Anderson et al. 2017); however, their success depends on the status of both the NLT and NPV, as applying GnRHa while NPV GnIH activity is high can lead to oocyte atresia (Anderson et al. 2017). Monitoring GnIH levels via qPCR or immunoassays may improve spawning synchrony by predicting optimal treatment timing, as low GnIH titers in Nile tilapia have been shown to predict optimal responsiveness to GnRHa treatment (Biran et al. 2014, Di Yorio et al. 2019, Li et al. 2022).

Na⁺/K⁺-ATPase distribution: a marker of neuronal activity and function

The widespread detection of the NKA α-subunit in neuronal cell membranes and neuronal fibers across multiple brain regions is a key finding of this study. NKA is fundamental to the maintenance of ionic gradients across neuronal membranes, which are essential for establishing resting membrane potential, generating action potentials, and supporting synaptic transmission (Clarke et al. 2013). The NKA pump actively transports Na⁺ and K⁺ ions against their concentration gradients, establishing and preserving the electrochemical conditions necessary for proper neuronal function (Wang and Huang 2006). The presence of NKA may therefore indicate regions of high neuronal activity and metabolic demand. The absence of NKA immunoreactivity in the telencephalon, despite the presence of neurons and blood vessels in this region (Fig. 3A-C), is consistent with previous observations in related species (Senarat et al. 2016, 2020) and may reflect differential expression levels, reduced antibody accessibility in the specific tissue preparation used, or the expression of region-specific NKA isoforms.

NKA detection in the optic tectum supports the high energetic demands of visual processing, while its presence in cerebellar Purkinje cells and medullary neurons is consistent with the high-frequency firing rates of these cells, which are essential for fine motor control and autonomic functions (Zhang et al. 2011). Further molecular investigations of NKA gene expression and neurotransmitter receptor distribution are needed to fully elucidate the physiological role of NKA in the fish brain (Shrivastava et al. 2019).

Melanomacrophage centers in the brain: implications for health assessment

The presence of MMCs within the brain parenchyma, particularly in connective tissue adjacent to blood vessels, the NLT, and the saccus vasculosus, is a notable finding of this study. While MMCs are well-established biomarkers of immune response, stress, and environmental contamination in hematopoietic organs of teleosts (Agius and Roberts 2003, Galindo-Villegas and Hosokawa 2004, Steinel and Bolnick 2017, Viana et al. 2021), their occurrence within the brain has rarely been documented. The NLT is a known integrator of stress responses and reproductive control (Li et al. 2022, Di Yorio et al. 2019), and the localization of MMCs near these critical neuroendocrine sites in all sampled fish may indicate a localized immune or stress response within the central nervous system. Given that the sampled fish were from a captive rearing environment, the presence of MMCs may reflect chronic or subclinical stressors such as water quality variation, stocking density, or handling. This finding raises the possibility of using brain MMCs as a sensitive early indicator of stress, potentially more informative than MMC assessments in peripheral organs. However, MMC abundance can also be influenced by age and diet (Matsche et al. 2023, Phromkunthong et al. 2015), and baseline MMC levels in healthy wild S. lysan brains would be required for meaningful comparison.

This study provides the first integrated neurobiological assessment of S. lysan and lays the groundwork for future research. Comparative studies between wild and captive populations are needed to determine whether the features observed here - particularly the prevalence of MMCs - are inherent to the species or induced by rearing conditions. Further investigations using molecular tools to quantify the expression of stress-related genes, such as those encoding GnIH or cortisol receptors, within the NLT and NPV are necessary to directly correlate environmental parameters with reproductive inhibition. This foundational knowledge is indispensable for developing sustainable aquaculture practices that address the biological and welfare needs of S. lysan and improve both productive efficiency and health in captivity.

Data Availability Statement

All data generated and/or analyzed are included in this article.

ACKNOWLEDGEMENTS

We thank the Department of Marine Science, Rajamangala University of Technology Srivijaya (Trang Campus), and the Microtechnique Laboratory (MIC-LAB), Division of Biological Science, Faculty of Science, Prince of Songkla University, for technical assistance. The authors would like to express their sincere gratitude to Dr. Natthawut Charoenphon and Ms. Kitiya Kongthong for their valuable assistance with the photography of brain specimens.

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• https://zoobank.org/6EC7ECE5-A4CF-4586-9DD9-60FDF181831C
• Funding Statement
  This research was partially supported by Chiang Mai University, Thailand.
• Ethical Statement
  All animal-related procedures were approved by the Animal Care and Use Committee of Rajamangala University of Technology Srivijaya (approval ID: IAC 13-12-64).
• AI Statement
  Artificial intelligence tools were used solely to assist with language editing and grammar.
• How to cite this article
  Phinrub W, Tanyaros S, Thaochan N, Nganvongpanit K, Lida A, Sornying P, Kenthao A, Senarat S (2026) Neuroanatomy, Na⁺/K⁺-ATPase distribution, and melanomacrophage centers in the brain of pond-reared female doublespotted queenfish, Scomberoides lysan (Perciformes: Carangidae). Zoologia 43: e25095. https://doi.org/10.1590/S1984-4689.v43.e25095
• Published by
  Sociedade Brasileira de Zoologia at Scientific Electronic Library Online - https://www.scielo.br/zool

Edited by

• Editorial responsibility:
  Carolina Arruda Freire

Publication Dates

• Publication in this collection
  07 Sept 2026
• Date of issue
  2026

History

• Received
  10 Oct 2025
• Accepted
  27 Jan 2026