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. 2026 Mar 6;109(1):111–129. doi: 10.1111/jfb.70366

Microanatomical characterization and histochemistry of the Siamese dwarf rasbora, Trigonostigma somphongsi (Meinken, 1958), a critically endangered species in Thailand: Implicating to the conservative biology

Tappadit Mitparian 1, Sitthi Kulabtong 2,3, Narit Thaochan 4, Atsuo Iida 5, Koraon Wongkamhaeng 6, Chavalit Vidthayanon 7, Korakot Nganvongpanit 8, Anan Kenthao 9,10, Sinlapachai Senarat 11,12,✉
PMCID: PMC13397249  PMID: 41789784

Abstract

The fundamental organization of fish tissue is crucial to bridge the fields of histopathology and physiology throughout research on biology. This study examines tissue examination of the Siamese dwarf rasbora, Trigonostigma somphongsi (Meinken, 1958), a critically endangered freshwater fish in Thailand. All fish were obtained from the freshwater area in Nakhon Nayok Province, Thailand, and were then processed using the standard histological and histochemical methods. Our observation clearly provides a detailed histological pattern of the major systems. Interestingly, the oral cavity had three distinct regions based on the presence of a taste bud, a mucous secreting cell and a longitudinal fold in the anterior, middle and posterior regions, referring to the digestive function. Moreover, the mature gonads (mature oocytes and spermatozoa) from all fishes indicate the adult stage. It is noted that this is a foundation of reproductive success under a biological life history, which is implicated to protect the area‐based conservation during the rainy season. This study uses a histological approach to provide information related to the systematic functional and anatomical phenomenon of T. somphongsi throughout the basic information for the effective area‐based conservation.

Keywords: critical freshwater area, histology, reproductive biology, the Siamese dwarf rasbora, Thailand

1. INTRODUCTION

Global biodiversity is declining at an unprecedented rate, creating an urgent need to understand and protect critically endangered species, especially those confined to specific geographical areas with unique habitat requirements (Kottelat & Witte, 1999; Vidthayanon, 2011). The Siamese dwarf rasbora, Trigonostigma somphongsi (Meinken, 1958), represents a distinctive part of Thailand's freshwater biodiversity. This small cyprinid species has specific habitat requirements and has become a key focus for conservation efforts due to its extremely limited distribution found solely within the Mae Klong, Chao Praya and Bangpakong basins (Kottelat & Witte, 1999; Petsut et al., 2014). The species was remarkably rediscovered in 2006 after being presumed extinct for two decades, which underscored both its resilience and vulnerability (Petsut et al., 2014). Due to its limited distribution, rarity in nature and specific habitat preferences, this species is classified as a critically endangered fish under criteria A2ac (Vidthayanon, 2011) and is listed as one of the world's 100 most threatened species (Baillie & Butcher, 2012). Given the increasing pressures from habitat degradation, pollution and climate change, there are growing concerns about the sustainability of wild T. somphongsi populations. A full understanding of the DNA barcoding (Panprommin & Panprommin, 2017) and habit distribution (Petsut et al., 2014) has been reported; however, knowledge of the anatomy and physiology of this species remains limited.

The successful conservation of endangered fish species requires detailed knowledge of their physiological adaptations and tissue‐specific responses to environmental stressors. Histological examination serves as a powerful tool for understanding these adaptations, particularly in species facing severe habitat pressures (Budi et al., 2025; Genten et al., 2009). Recent studies have demonstrated how histological insights can directly inform conservation strategies, such as identifying optimal environmental conditions for reproduction (Alonso‐Fernández et al., 2011; Griffen & Weber, 2025; Lowerre‐Barbieri et al., 2023; Mayer & Pšenička, 2024) and assessing tissue‐level responses to habitat degradation (Bernet et al., 1999; Liebel et al., 2013). Detailed histological analyses can shed light on the intricate cellular compositions and architectural arrangements within the organs, facilitating a deeper comprehension of their roles and potential susceptibilities to environmental stressors or pathogenic agents (Genten et al., 2009; Heins & Brown‐Peterson, 2022). For critically endangered species like T. somphongsi, such information becomes crucial for developing effective conservation protocols and captive breeding programmes.

To date, the histological attributes of T. somphongsi remain largely unexplored, with current knowledge primarily derived from broader ecological observations and aquaculture practices. Nowadays, the sampled fish is also increasingly exported as freshwater ornamental aquarium fish, which may correspond to its population decline (Petsut et al., 2014). This knowledge gap therefore obstructs the establishment of effective conservation strategies and the development of optimal husbandry practices. The present study aims to address this deficiency by conducting the first comprehensive histological and histochemical analysis of T. somphongsi's vital organs and tissues. We hypothesize that understanding the species’ microanatomical characteristics will reveal specific adaptations that could inform both in situ conservation efforts and ex situ breeding programmes. This research seeks to establish a baseline histological atlas that can serve as a reference for future health assessments of wild populations and guide the development of species‐specific conservation protocols.

2. MATERIALS AND METHODS

Twenty mature, healthy individuals of T. somphongsi (10 females, 2.3 ± 0.9 cm total length and 10 males, 2.2 ± 0.5 cm total length) without their parasitic appearances and lesions were sampled by beach seine from the freshwater area in Nakhon Nayok Province, Thailand (14° 5′ 24.32″ N and 101° 16′ 52.93″ E) during the rainy season (September–November). All animal procedures were ethically and methodologically approved by the Animal Care and Use Committee of the Rajamangala University of Technology Tawan‐ok, with the ID #RMUTTO‐ACUC‐2‐2022‐0011. During fieldwork, all collected fish were killed by a rapid cooling shock (Wilson et al., 2009), and their whole body was fixed in 10% neutral buffer formalin (NBF) for 24 h at room temperature. Then, the fixed fish were kept in 70% alcohol for taxonomic confirmation and histological observations. The identification of the sampled fish species was confirmed using the publication of Meinken (1958).

For histological observations, the fixed samples were dehydrated in a series of increasing ethanol concentrations (70%, 95% and 100%), cleared in xylene and embedded in paraffin using standard histological techniques (Presnell & Schreibman, 1997; Suvarna et al., 2013). The paraffin‐embedded blocks of whole fish body were then cut into 4‐μm‐thick sections in both longitudinal and transverse plans. The unstained sections were stained with Harris's haematoxylin and eosin (H&E) to observe the normal structure and cell types through the histochemically stained with Periodic acid‐Schiff (PAS), Masson's Trichrome and Reticulin staining methods (Presnell & Schreibman, 1997; Suvarna et al., 2013). The stained histological slides were examined to observe the structur‐e and the reproductive features in relation to the total length. All data were finally photographed using a 3DHISTECH Panoramic Viewer (3DHISTECH, Hungary).

3. RESULTS

3.1. Female reproductive system

The female reproductive structure and the oogenesis are presented in Figure 1. This system comprises the ovary and a reproductive duct (Figure 1a–d). The ovary containing differentiating oocytes is in the peritoneal cavity and is closely associated with several internal organs, such as the swim bladder and intestine (Figure 1a,b). The oogenesis of all mature studied samples is a highly organized composition of oocytes and is traditionally divided into three distinct phases, including the primary oocyte (Po), secondary oocyte (So) and mature oocyte (Mo) phases (Figure 1c). Based on the growth pattern of the oocytes, the ovary undergoes asynchronous development (Figure 1c). The mature oocytes are released into the oviduct (Figure 1d).

FIGURE 1.

FIGURE 1

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Light microscopic level of the female reproductive system of Trigonostigma somphongsi. (a) The localization of ovary (ov) in the peritoneal cavity. (b–d) The oval organ of female reproductive structure (black dash line) with the prominent of oocytes (oc) dividing into primary growth stage pg), secondary growth stage (sg) and mature oocyte (mo). (e) Two stages of pg: (i) the perinucleolar stage (ps) with a central and round nucleus (n) and a strongly basophilic ooplasm surroundings with the follicular cell (fc) and (ii) the cortical alveoli and oil droplet stage (oac) with the prominent of cortical alveoli (ca) and oil droplet (od). (f) The early secondary growth stage (esg) with initiation of yolk granule accumulation (yg). (g) The positive reaction +PAS in both cortical alveoli (ca) and yolk granule (yg) in the esg. (h) The initially developed micropyle (mp). (i) The late secondary growth stage (lsg) with the great accumulation of yolk granules (yg). (j) The mature oocyte (mo) with addressing the formation of yolk plate (yp). br, brain; cf, caudal fin; df, dorsal fin; es, oesophagus; fc, follicular cell; gi, gill; ki, kidney; in, intestine; ms, muscular skeleton; n, nucleus; nu, nucleolus; oca, oral cavity; red, reproductive duct; sb, swim bladder; to, tongue; zp, zona pellucida. Staining methods: (a, c–f, h–j) Harris's haematoxylin and eosin (H&E); (b) Masson's Trichrome staining method; (g), Periodic acid‐Schiff reaction.

3.1.1. Primary oocyte growth (pg)

Oocytes in the pg stages were categorized into two distinct stages: the perinucleolar stage (ps) and the oil droplet and cortical alveoli stages (oac). The ps has an oval shape with a central, round nucleus and several nucleoli near the nuclear membrane (Figure 1e). This cell has a strongly basophilic cytoplasm and is surrounded by follicular cells (H&E staining method, Figure 1e). After the Ps develops into the oac, it increases in size due to the accumulation of oil droplets and cortical alveoli (Figure 1e). An oval oil droplet and slightly basophilic cortical alveoli initially appear close to the nuclear membrane (Figure 1e). During folliculogenesis, the follicular complex differentiates and subdivides into two layers: zona pellucida, which is a thin cellular acidophilic layer, and the follicular cell layer (Figure 1e).

3.1.2. Secondary oocyte growth (sg)

During the growth of the sg, there are two start‐up stages: early (esg) and late (lsg) secondary growth stage (Figure 1f,h). The Esg oocyte is easily recognized by an increase in size due to an acidophilic small yolk granule with PAS+ located close to the follicular complex, which is a well‐conserved aspect in reproductive biology (Figure 1f,g). The initially developed micropyle exists (Figure 1h). At the stage of the follicular complex, there is continuous development, especially in the zona pellucida (Figure 1f).

The lsg oocyte is abundant in the ovarian tissue and appears to increase in size compared to the previous stage (Figure 1i). Furthermore, an accumulation of many yolk granules of varying sizes can be found throughout the ooplasm (Figure 1i).

3.1.3. Mature oocyte (mo)

The Mo is the largest in size. The ooplasm is completely filled with enormous yolk globules, resulting in the formation of the yolk plate (Figure 1j).

3.2. Male reproductive system

The testes of T. somphongsi are located close to the kidney and swim bladder (Figure 2a). It is a paired organ with a series of seminiferous tubules (Figure 2b,c). The segments of spermatogenic tubules in all observed samples comprise several stages of sperm development, which can be classified into five stages, from spermatogonium to spermatozoon (Figure 2b,c). Spermatogonia are located close to the basement membrane and have an oval shape and a large eosinophilic nucleus (Figure 2d). During the spermatogenetic process, spermatogonia develop into primary and secondary spermatocytes, respectively. The primary oocytes are large cells with eosinophilic cytoplasm and a distinct nucleus containing dense chromatin. The secondary spermatocytes are small, spherical cells with a spherical nucleus and filamentous chromosomes (Figure 2e). Spermatids have an oval shape, a large nucleus and a small amount of cytoplasm. Some cells begin to develop a tail structure at this stage (Figure 2f). Mature spermatozoa are released into the lumen, and each cell can be distinguished by two features: a basophilic oval head and an eosinophilic tail (Figure 2g).

FIGURE 2.

FIGURE 2

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Light microscopic level of the male reproductive system of Trigonostigma somphongsi. (a) Testicular organ (tes) of T. somphongsi were close to the kidney (ki). (b–g) Several seminiferous tubules consisting of spermatogenic stages including spermatogonia (sg), primary spermatocytes (ps), secondary spermatocytes (ss), spermatids (st) and spermatozoa (sz). High magnification revealing an oval shape and a large eosinophilic nucleus (n) of spermatogonium (sg) (d), primary (ps) and secondary (ss) spermatocytes (e), the initiation of the tail structure (tai) of spermatids (st) and the basophilic oval head (he) and eosinophilic tail (tai) of spermatozoa (sz). br, brain; cy, cytoplasm; ey, eye; gi, gill; oo, olfactory organ; sb, swim bladder. Staining methods: (a–g) Harris's haematoxylin and eosin.

3.3. The digestive system and accessory digestive organs

The digestive system of T. somphongsi is composed of the digestive tract (from the oral cavity and oesophagus to the intestine) and accessory organs such as the pancreas and liver, as illustrated in Figures 3 and 4.

FIGURE 3.

FIGURE 3

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Light microscopic level of the digestive system and accessory digestive organs of Trigonostigma somphongsi. (a) The feature of digestive tract was divided into mouth having three regions including anterior (1), middle (2) and posterior (3) regions, pharynx (pha), oesophagus (es) and intestine (in) throughout the localization liver (li) and pancreas (pa). (b) The upper jaw covering an oral epithelium (oe) and the development bone structure like teeth (bo). (c) The lower jaw having a bone structure like teeth (bo) appearing close to the skeletal muscle (smu). (d–f) High magnification showing the oral epithelium, mucous secreting cells (msc) and taste buds (tb). (g) The middle region (2) and posterior region (3) with the prominent of pharynx (pha) and the well‐defined long folded structure (arrows). (h–k) The stratified oral epithelium (ep) lining on the basement membrane (**) throughout the +PAS positive reaction of mucous secreting cells (msc). (l, m) The tongue lining with an oral epithelium (oe), the connective tissue (cnt) and the supporting hyaline cartilage (hy). (n, o) The presence of stratified pharynx epithelium (4) covering the horny pad (5). (p) The well development of mature teeth with dentine (de) was classified in close to the mucous secreting cells (msc). *, basement membrane; at, atrium; cb, cerebrum; cn, cerebral hemisphere; cnt, connective tissue; ig, integument; ki, kidney; md, medulla oblongata; ms, muscular skeleton; msc, mucus secreting cells; ob, olfactory bulb; oca, oral cavity; ot, optic lobe; ov, ovary; sb, swim bladder; smu, skeleton muscle; spc, spinal cord; sv, sinus venosus; to, tongue; vl, vagal lobe; ve, ventricle; arrows, the projection of connective tissue together with the reticular fibre. Staining methods: (a, b, d, g, h, l, n, p) Harris's haematoxylin and eosin, (c, e, i, m) Masson's Trichrome method, (g, k) Periodic acid‐Schiff reaction, (j, o) Reticulin staining method.

FIGURE 4.

FIGURE 4

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Light microscopic level of the digestive tract of Trigonostigma somphongsi. (a) The junction between pharynx (pha) and oesophagus (es). (b) Four distinct layers including the mucosa (mu), submucosa (sm), muscularis (mus) and serosa (data not shown). (c) The anterior intestine (ai) with numerous high‐longitudinal folds (lf). (d) The posterior intestine (pi) with the low longitudinal fold (lf) and the prominent goblet cell (gc). (e–g) High magnification showing the columnar epithelial epithelium (ep) and specialized goblet cells (gc). (h) The pancreas with the acini (ac) and blood vessel (bv). (i, j) The hepatic parenchyma (hpa) with the hepatocytes (hc) and hepatic sinusoids (si). (k) High magnification showing the hepatocytes with a concentric nucleus and oil droplet (oi) in the cytoplasm. ep, epithelium; lp, lamina propria; ms, muscularis; mu, mucosa; msc, mucous secreting cell; se, serosa; arrows, basement membrane. Staining methods: (a–c, e, h, k) Harris's haematoxylin and eosin (H&E), (b, g, i) Masson's Trichrome, (d, f, j) Reticulin staining method.

3.3.1. Oral cavity

The oral cavity acts as the link between the gill respiratory chamber and the digestive tract and can be categorized into three regions: anterior, middle and posterior (Figure 3a). The cavity is lined with stratified squamous epithelium, and bone structures such as teeth develop inside the connective tissue in both the upper (Figure 3b) and lower jaws (Figure 3c). Several taste buds and mucous‐secreting cells were discovered in the stratified oral epithelium lining on the basement membrane (Figure 3d–f). The epithelial lining on the thick basement membrane of the posterior regions is thicker than the anterior region (Figure 3g). The presence of taste buds and mucous‐secreting cells is evident (Figure 3h–j), with the mucous‐secreting cells showing positive staining for the PAS+ method in their granules (Figure 3k). Additionally, this region contains a slightly developed tongue made up of oral epithelium, connective tissue and supporting hyaline cartilage (Figure 3l,m). Compared to the posterior region, its upper feature is composed of a stratified pharynx epithelium covering the horny pad (Figure 3n). At the upper zone, the projection of connective tissue and the reticular fibre are easily identified (Figure 3n,o). On the lower side, the well‐defined long folded structure (Figure 3g) and the well‐developed mature teeth with dentine were classified as close to the MSC (Figure 3p).

3.3.2. Oesophagus

The oesophagus is continuously formed by the pharynx (Figure 4a). Its wall has four distinct layers: the mucosa, submucosa, muscularis and serosa (Figure 4b). The mucosal layer displays significant folding and is covered with simple squamous epithelial cells interspersed with oval mucus‐secreting cells (Figure 4b). In the submucosa, there are loose and dense connective tissues with greenish tones when stained using the MT method (Figure 4b). The prominent smooth muscle of the muscularis, with a dark red tone using the MT staining method, is typically present (Figure 4b).

3.3.3. Intestine

The intestine of T. somphongsi consists of one long tube in the abdominal cavity, which is divided into two regions: the anterior (Figure 4c) and the posterior (Figure 4d) intestines. The anterior intestine begins with the oesophagus and has numerous high‐longitudinal folds (Figure 4c). The mucosa of the anterior intestine is lined with columnar epithelial epithelium and specialized goblet cells (intestinal goblet cells) on the thickness of the basement membrane (Figure 4e–g). The posterior intestine shares similar histological features with the anterior intestine, although it exhibits low longitudinal folds with prominent goblet cells (Figure 4d).

3.3.4. Digestive accessory organs (pancreas and liver)

The digestive glands, that is, liver and pancreas, play a crucial role in gastric digestion in T. somphongsi because they lack gastric gland secretion. The pancreatic tissue contains various components, particularly the exocrine acini consisting of pyramidal acinar cells and numerous blood vessels (see Figure 4h). The liver tissue (or hepatic parenchyma) consists of hepatocytes, hepatic cords lined with reticulin and hepatic sinusoids (Figure 4i,j). The hepatocytes are irregular in shape, and concentric nuclei are also observed (Figure 4k). In addition, some hepatocytes contain a large oil droplet (Figure 4k).

3.4. Respiratory system

3.4.1. Gills

The gill is located on the side of the head, extending from the floor to the roof of the buccal cavity (Figure 5a). It is a four‐paired structure, with each pair formed by a network of three complete components, including the gill arch, gill raker and double rows of gill filaments (Figure 5b).

FIGURE 5.

FIGURE 5

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Light microscopic level of the respiratory system of Trigonostigma somphongsi. (a) The localization of gill (gi) and pseudobranch (pb). (b) The gill raker (gr), gill arch (ga) and gill filament (gf) of gill. (c) The gill raker lining with the stratified epithelium (ep), mucus secreting cell (msc) and teeth containing the bone structure (bo) was observed, whereas the gill arch had a cartilaginous supporting structure (hy) and the afferent branchial arteries (art). (d) The primary (pl) and secondary (sl) lamellae of gill filament. (e, f) High magnification showing the gill erythrocyte (ec), pillar cell (pc) and chloride cells (cc). (g, h) The pseudobranch with the pseudobranchial lamella (pl) containing the capillaries (cp) and the glandular pseudobranch cell (arrows). bv, blood vessel; hy, hyaline cartilage; sb, swim bladder. Staining methods: (a, d) Masson's Trichrome method, (b, c, e–g) Harris's haematoxylin and eosin.

The gill raker is the structure projecting from the gill arch (Figure 5b). It is lined by stratified epithelium, which includes the mucus‐producing cells and osseous teeth‐like projections in the inner edge (Figure 5c). The gill arch is supported by bone and hyaline cartilage (Figure 5c). The branching patterns of the afferent branchial arteries in this region were identified (Figure 5c).

Each gill filament is divided into primary and secondary lamellae (Figure 5d). The primary lamella is attached to the gill arch and supported by a thin central hyaline cartilage (Figure 5d) and the blood vessel (Figure 5e). At high magnification, the stratified epithelium and chloride cells are found at the base of the primary lamella (Figure 5f). Additionally, the prominent oval‐shaped nucleus of the epithelial cells is easily identified in the interlamellar epithelium (Figure 5d). The secondary lamellae are hair‐like structures that originate from the upper and lower surfaces of primary lamellae and are oriented perpendicular to the filaments (Figure 5d). They are covered with a single layer of squamous epithelium, also called ‘the respiratory epithelium’ (Figure 5e). Erythrocyte and oval contractile pillar cells are present between enriched blood spaces in the secondary lamellae (Figure 5e).

3.4.2. Pseudobranch

The pseudobranch, a gill‐like structure, is differentiated from the first gill arch (Figure 5a). This organ has several rows of parallel lamellae (Figure 5g). It is covered by a thick layer of secondary pseudobranchial lamellae containing capillaries and glandular pseudobranchial cells (Figure 5h). The glandular pseudobranch cells, or pseudobranchial cells, are easily identifiable and located between the lamellae (Figure 5h). Each cell has an oval shape with a central nucleus and clear cytoplasm, which exhibits a slightly acidophilic feature (Figure 5h).

3.5. Nervous system

3.5.1. Brian

We provide an overview of the telencephalon that contains the cerebral hemisphere and olfactory bulb, which are associated with the olfactory tract (Figure 6a–c). A strongly conserved cell taxonomy between these areas is identified. Neuroglial diversity, neuronal fibre and blood vessels are presented in Figure 6d,e.

FIGURE 6.

FIGURE 6

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Light microscopic level of the nervous system of Trigonostigma somphongsi. (a, b) Overall brain in longitudinal section from the anterior to posterior views including olfactory bulb (ob), cerebral hemisphere (ch), optic lobe (ot), hypothalamus (hyp), pituitary gland (py), valvuli cerebelli (vc), corpus cerebelli (cc), vagal lobe (vl), medulla oblongata (md). (c) The olfactory bulb (ob) liking with the olfactory tract (ofl). (d) The neuroglial population (arrows) in the olfactory bulb. (e) The cerebral hemisphere consisting of neuroglial diversity (arrows) and blood vessel (bv). (f, g) A highly organized structure of optic tectum including stratum marginale (1), stratum opticum (2), stratum fibroetgricialem (3), stratum album central (4), stratum griseum central (5) and stratum periventriculae (6). (h) The corpus cerebelli. (i) The valvula cerebelli. (j) The neurons (circled) and neuroglia (arrows). (k) The large area of hypothalamus (hyp). (l) The adenohypophysis (ad) neurohypophysis (ne) of the pituitary gland. epit, epithalamus; is, infundibular stalk; noc, notochord; scc, semicircular canal; spc, spinal cord; ml, molecular layer; pc, Purkinje cell layer; gl, granular layer; th, thalamus; *, third ventricle. Staining methods: (a, l) the Periodic acid‐Schiff reaction (PAS), (b–f, h, j, k) Harris's haematoxylin and eosin (H&E), (c, g) Reticulin staining method.

The well‐developed optic tectum (or the mesencephalic optic tectum) has a highly organized structure with multiple layers, including stratum marginale, stratum opticum, stratum fibroetgricialem, stratum album central, stratum griseum central and stratum periventriculae (Figure 6f,g).

The cerebellum is the main region of the metencephalon, which is posterior to the brainstem and positioned beneath the optic tectum (Figure 6b). Herein, it can be subdivided into two sub‐regions including corpus and valvula cerebelli (Figure 6b). A tightly folded structure of cerebelli regions, especially corpus cerebelli, is exhibited, but all structural regions are shared. Three distinct layers, from outer to inner layers, include the outer molecular layer with a densely packed small granule cells, the specialized Purkinje cell layer with its small pyramidal cell and the inner granular layer with information flowing through the specialized neuronal cells which are remarkably arranged (Figure 6h–j). It is noted that the detailed overview of Purkinje cell had similar fractal structures along the regions. It has a large pear shape and an elongated axon (Figure 6i).

The diencephalon is made up of three zones, from the dorsal to ventral views, including the dorsal epithalamus as a small portion, the middle thalamus and the ventral hypothalamus line along the wall of the third ventricle (Figure 6k).

The pituitary gland, a glandular tissue, is a triangular‐shaped structure located in the sella turcica, which is connected to the hypothalamus by the infundibular stalk (Figure 6l). Figure 6l presents adenohypophysis, which demonstrates the cell clusters and a positive reaction to PAS and the neurohypophysis of this organ.

3.5.2. Spinal cord

The spinal cord is a tubular structure that is continuous from the myelencephalon (Figure 7a). The arrangement of this organ is formed by grey matter and white matter (Figure 7a). The grey matter is mainly made up of a specialized population of neurons and neuroglia (Figure 7a), whereas the white matter structure contains a network of the myelinated nerve fibres (Figure 7a).

FIGURE 7.

FIGURE 7

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Light microscopic level of the spinal cord and notochord (noc) of Trigonostigma somphongsi. (a) The spinal cord (spc) with grey matter (gm) and white matter (wm). (b) The localization of notochord close to the cord. (c) The bone matrix (bma) around the notochord. (d) Large irregularly shaped notochord vacuolated cells (*) as an eccentric nucleus (enu). nf, neuronal fibre; neu, neurons; ng, neuroglia. Staining methods: (a) Harris's haematoxylin and eosin; (b–d), Periodic acid‐Schiff reaction.

3.5.3. Notochord

The notochord is located within the spinal column in parallel to the spinal cord (Figure 7b). Large irregularly shaped notochord vacuolated cells as an eccentric nucleus are observed and surrounded by the bone matrix (Figure 7c,d).

3.6. Integument

Our observations show that the integument, with many histologically identified features (Figure 8a), can be classified into two layers, including the epidermis and dermis (Figure 8a).

FIGURE 8.

FIGURE 8

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Light microscopic level of the integument of Trigonostigma somphongsi. (a) Two layers including the epidermis (epi) and dermis (der) in the integument (ig). (b, c) There were three layers including the stratum basale (sba), the fusiform layer (fl) and the cuticular layer (cla). Several mucous secreting cells (msc), Malpighian cell (mac) and epidermal cell (ec) in the fusiform layer. (d) +PAS reaction with the mucous secreting cell (msc). bm, basement membrane; cnt, connective tissue; sca, scale. Staining methods: (a, c) Masson's Trichrome method, (b) Harris's haematoxylin and eosin, (d) Periodic acid‐Schiff reaction.

3.6.1. Epidermis

The fundamental arrangement of epidermis covers the outer surface of the body, forming three distinct layers, which from inner to outer regions include the stratum basale (or germinativum), the fusiform layer (or middle layer) and the cuticular layer as a thin non‐cellular external region (Figure 8b). The stratum basale is a single row of cuboidal basal epithelial cells above a thin basement membrane (Figure 8b). A multilayered layer of fusiform is normally thicker than in many other species due to the abundance of the cells, including the mucous secreting (or goblet) cells (msc) and an oval shaped Malpighian (or epidermal) cell (MC) (Figure 8b,c). It is noted that the vacuolated msc consists of unicellular intraepithelial mucin‐secreting glands among the MC (Figure 8b,c), but it reacted with the PAS‐pinkish positive material (PAS staining method) (Figure 8d).

3.6.2. Dermis

The location of the dermis is below the epidermis (Figure 8a), formed by elaborate arrays of connective tissue layers (Figure 8c). The deeply embedded scale was identified (Figure 8c).

3.7. Circulatory systems

The heart of the sample is caudoventrally located in the head and ventrally situated in the isthmus (Figure 9a). The position of its organ was observed within the pericardium of the thoracic cavity (Figure 9a), in relation to the gill cavity (Figure 9a). The sampled heart is composed of four distinct including the sinus venosus, atrium, ventricle and bulbous arteriosus (Figure 9a).

FIGURE 9.

FIGURE 9

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Light microscopic level of the circulatory systems of Trigonostigma somphongsi. (a) The heart consisting of four chambers including the sinus venosus (sv), atrium (at), ventricle (ve) and bulbus arteriosus (ba). (b) The epicardium (epic) and endocardium (end) of sinus venosus. (c) The junction between ventricle, sinus venosus (sv), ventricle (ve) and bulbus arteriosus (ba). (d) The myocardium (or cardiac muscle tissue, cmt) in the atrium. (e, f) The myocardium with the cardiac muscle tissue (cmt) and the reticular fibre (arrows). (g, h) The wall of the bulbous arteriosus (*) with the prominent connective tissue (**) and reticular tissue (double arrows). cs, coronary sinus; erc, erythrocytes; oca, oral cavity; gi, gill; gic, gill cavity. Staining methods: (a–d, g) Periodic acid‐Schiff reaction, (e) Masson's Trichrome method, (f) Reticulin staining method.

The sinus venosus is a thin‐walled cavity along with the left hepatic vein drainage to the coronary sinus, which is the caudal chamber of the heart (Figure 9a). It was difficult to identify the wall of this chamber through histological observation, but there were two layers (the epicardium and endocardium) (Figure 9b). The erythrocytes were widely accumulated (Figure 9b).

The atrium is posteriorly linked with the sinus venosus (Figure 9a). However, the myocardium (or cardiac muscle tissue) was first observed, compared to the previous chamber (Figure 9c,d). Meanwhile, the ventricle with prominent myocardium was seen (Figure 9e). This structure basically contained the reticular fibre (Figure 9f).

The wall of the bulbous arteriosus is mainly composed of connective tissue and reticular tissue (Figure 9g,h) and supported by the blood cell and erythrocyte (Figure 9g).

3.8. Excretory system

The kidney structure of T. somphongsi is a pair of bilateral organs placed retroperitoneally close to the vertebral column (Figure 10a).

FIGURE 10.

FIGURE 10

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Light microscopic level of the urinary system of Trigonostigma somphongsi. (a) The kidney with diving to anterior (ak) and posterior (pk) kidney. (b, c) The haematopoietic tissue (ht) and blood cell formation as well as the blood cell population (bcp) in the anterior kidney. (d) Some cells of blood cell population (bcp) with + PAS method. (e, f) The posterior kidney with the prominent of nephrons with a glomerulus (go), proximal convoluted tubules (pc) and distal convoluted tubules (dc). (g) A tall columnar epithelium (ep) with the brush border (*). es, oesophagus; ht, haematopoietic tissue; vc, vertebral column. Staining methods: (a, b, e, f) Harris's haematoxylin and eosin, (c) Masson's Trichrome method, (d, g) Periodic acid‐Schiff reaction.

Based on the location and histological structure, the sampled kidney is classified into anterior and posterior kidneys (Figure 10a). Detailed information concerning the anterior kidney is that it is closely located in the oesophagus, but only composed of haematopoietic tissue and blood cell formation which are described in Figure 10a–c. Some cells reacted positively with the PAS method (Figure 10d).

The posterior kidney has nephrons with a glomerulus, proximal convoluted tubules and distal convoluted tubules (Figure 10e–f), which were found from the middle (Figure 10e) to posterior (Figure 10f) localization. In terms of the proximal convoluted tubule, it is covered by a tall columnar epithelium with basal nuclei (Figure 10g).

4. DISCUSSION

Documenting the normal histology of fish tissues and organs provides a basis to expand our knowledge of fish pathology (Genten et al., 2009). Histological examination can help identify signs of disease that may not be visible through gross anatomy examination, making it valuable for monitoring fish health (Bernet et al., 1999; Zimmerli et al., 2007). Moreover, histological analysis of fish has been applied in various aspects, such as biomarkers of water quality or pollutant contamination (Bernet et al., 1999; Liebel et al., 2013; Yancheva, 2016), the study of reproductive biology (Alonso‐Fernández et al., 2011; Heins & Brown‐Peterson, 2022), the diagnosis of fish diseases (Chinchilla et al., 2023; Feist & Longshaw, 2008; Gjessing et al., 2019), as well as the evaluation of feeding meals and rearing conditions on the development and growth of aquaculture fishes (Bonvini et al., 2018; Matulić et al., 2020). The present study offers the first histological description of some major organs in T. somphongsi, including the digestive, reproductive, circulatory and integumentary systems. The results are consistent with the findings of previous studies above, yet some notable histological characteristics of T. somphongsi have been discovered.

The histological organization of tissues and organs provides fundamental insights into species adaptation and survival strategies (REF). The present study provides the first comprehensive characterization of T. somphongsi, offering distinctive structural adaptations in the digestive, reproductive and integumentary systems that distinguish it from cyprinid fishes. These findings address critical knowledge gaps while providing practical insights for conservation. The documented tissue‐level adaptations, particularly in the alimentary canal and reproductive organs, suggest specific environmental requirements that may explain both the species’ restricted distribution and its resilience. These baseline histological parameters establish essential references for population health assessment and conservation planning, especially crucial given the increasing anthropogenic pressures on T. somphongsi's natural habitat.

4.1. Digestive system adaptations and feeding ecology

The histological analysis of T. somphongsi's digestive system reveals significant modifications in the buccal cavity architecture, highlighting specialized adaptations for food detection and processing. These findings align with the broader diversity of gastrointestinal structures in fish, as reviewed by Wilson and Castro (2010), while showcasing unique features specific to T. somphongsi.

The buccal cavity exhibits distinct organizational patterns, including regions with specialized distributions of taste buds and mucous‐secreting cells. These structures suggest advanced gustatory capabilities, enabling efficient food detection and screening, as similarly noted in other cyprinids (Elsheikh et al., 2012; Yashpal et al., 2009). Notably, T. somphongsi possesses a filamentous epithelial projection and a spongy cushion‐like palatal organ in the posterior buccal cavity. This organ, also reported in families such as Cyprinidae, Catostomidae, Cobitidae and Salmonidae (Kapoor et al., 1976; Yashpal et al., 2009), facilitates the manipulation and propulsion of food particles towards the oesophagus (Callan & Sanderson, 2003). The lingulate epithelial projections of the palatal organ further enhance food transport efficiency, whereas the abundance of taste buds on these projections contributes to gustation, ensuring selective swallowing of food particles (Elsheikh et al., 2012). Additionally, the presence of a dentine teeth pad in the posteroventral oral cavity, coupled with pharyngeal teeth on the fifth ceratobranchial bones, reflects specialized adaptations for grinding and macerating food. These features compensate for the lack of oral jaw teeth, a characteristic trait in cyprinids, and underline the evolutionary versatility of T. somphongsi in optimizing feeding efficiency (Aigler et al., 2014; Pasco‐Viel et al., 2010).

The present study also illustrates that T. somphongsi lacks a true stomach, which is a common trait in the Cyprinidae family (Gonçalves et al., 2016; Kapoor et al., 1976), that has led to remarkable compensatory adaptations in intestinal structure and function. Our histological analysis reveals specialized modifications of the intestinal epithelium, characterized by prominent mucosal folds, an elaborate brush border and a high density of scattered goblet cells. These characteristics, resembling findings in other stomachless fish (Day et al., 2011; Leknes, 2014), indicate that the intestine functions both as a chamber for receiving undigested food particles and as the main site for enzymatic activity and nutrient absorption (Day et al., 2011; Gonçalves et al., 2016; Leknes, 2014).

Of particular significance is the differential distribution of goblet cells along the intestinal tract, with higher density in the posterior region compared to the anterior portion. This result aligns with findings observed in other cyprinids, such as Puntius stoliczkanus (Senarat et al., 2013). This organizational pattern likely reflects functional specialization, where increased mucus production in the posterior intestine enhances mucosal protection and lubrication, thereby facilitating faecal expulsion (Murray et al., 1996).

Interestingly, the long and specialized intestine with high mucosal folds throughout supports the hypothesis that the species is an omnivorous fish capable of processing cellulose‐rich diets over extended periods, as similarly found in Barbus stigma (Kapoor, 1957) and Culter alburnus (Cao et al., 2011). This adaptation may confer an ecological advantage, allowing T. somphongsi to exploit a broad range of food resources in its natural habitat (Leknes, 2014). These structural adaptations have important implications for understanding both the species’ dietary flexibility and its potential resilience to varying food resources in its threatened habitat.

4.2. Reproductive biology and conservation implications

Our histological analysis of T. somphongsi reveals sophisticated reproductive adaptations, with mature oocytes and spermatozoa present in specimens above 2.2 cm total length – establishing a critical biological benchmark. These findings confirm the sexual maturity of individuals in wild habitats (Heins & Brown‐Peterson, 2022). Moreover, T. somphongsi exhibits asynchronous ovarian development, characterized by oocytes at multiple developmental stages. Similar patterns of oocyte development have been observed by Rinchard and Kestemont (1996) in cyprinid fishes such as Rutilus rutilus, Alburnus alburnus and Blicca bjoerkna. Other studies have also reported similar findings in various fish species, including Gobio gobio (Linnaeus, 1758), Silurus microdorsalis (Mori, 1936) and Sperata aor (Hamilton, 1822). This asynchronous development represents a key adaptive strategy enabling the release of eggs in multiple batches over extended periods, potentially enhancing reproductive success under variable environmental conditions (Heins & Brown‐Peterson, 2022; Serrat et al., 2019).

The asynchronous development of oocytes, regulated by both physiological and environmental factors (Clelland & Peng, 2009; Kestemont, 1990; Ki & Lee, 2018), enables T. somphongsi to spawn multiple clutches rather than relying on a single spawning event. Such a strategy is advantageous in dynamic freshwater ecosystems, where fluctuating environmental conditions could impact reproductive timing. These findings emphasize the importance of further studies to document the reproductive season, spawning periods and egg production dynamics of T. somphongsi in its natural habitat.

The insights gained from this study have direct applications in both aquaculture and conservation. By establishing a detailed understanding of gametogenesis, this research supports the development of aquaculture strategies for T. somphongsi, including accurate reproductive phase assignments to optimize breeding programmes. Enhanced knowledge of the mechanisms underlying oocyte maturation and sperm development will improve fish production efficiency (Lowerre‐Barbieri et al., 2023).

From a conservation standpoint, the observed reproductive patterns of T. somphongsi provide essential information for key conservation measures. The size at sexual maturity at a total length above 2.2 cm provides critical baseline data for establishing size thresholds in conservation policies, whereas the asynchronous reproduction pattern suggests the need for extended protection periods. Understanding the relationship between environmental factors and reproductive timing enables more effective protected area designation. These findings, integrated with existing ecological data (Petsut et al., 2014) and genetic analyses (Panprommin & Panprommin, 2017), provide a comprehensive framework for evidence‐based conservation strategies. Effective conservation of T. somphongsi requires both temporal protection during peak reproductive periods and spatial protection of critical breeding habitats. Furthermore, integrating reproductive histology into ongoing monitoring programmes will allow for the assessment of population health and reproductive potential, ensuring the long‐term sustainability of T. somphongsi in the wild.

4.3. Physiological adaptations and environmental implications

The present study indicates that the respiratory system of T. somphongsi has specialized adaptations essential for survival in its restricted habitat. The highly vascularized gills of T. somphongsi, characterized by well‐developed primary and secondary lamellae, facilitate efficient gas exchange even in low‐oxygen environments (Gjessing et al., 2019; Liebel et al., 2013). The presence of chloride cells at the base of the primary lamellae suggests an additional role in osmoregulation, enabling the species to maintain ionic balance in freshwater habitats prone to fluctuating salinity or pollution levels (Genten et al., 2009; Liebel et al., 2013). Furthermore, the pseudobranch's distinctive structure, particularly its glandular pseudobranchial cells with clear cytoplasm and central nuclei, suggests specialized oxygen‐sensing capabilities (Bernet et al., 1999). This adaptation may be crucial for survival in habitats with variable oxygen levels (Zimmerli et al., 2007), potentially explaining the species’ habitat preferences and limited distribution (Petsut et al., 2014).

Moreover, T. somphongsi appears to have integumentary adaptations, which are characterized by three distinct layers of epidermis. These layers exhibit specialized features and are rich in mucous‐secreting cells, particularly in the fusiform layer. Their positive reaction to PAS staining suggests enhanced protective mechanisms against environmental stressors (Yancheva, 2016). This complex organization contributes to the species’ environmental resilience while requiring specific habitat conditions for optimal function (Bernet et al., 1999; Liebel et al., 2013).

These physiological adaptations have direct conservation implications, requiring maintenance of specific water quality parameters (Gjessing et al., 2019), prioritization of habitat protection matching physiological requirements (Petsut et al., 2014) and implementation of tissue‐based health indicators for environmental monitoring (Yancheva, 2016; Zimmerli et al., 2007). Understanding these specialized features provides a scientific basis for developing conservation strategies that consider both physiological requirements and environmental conditions (Vidthayanon, 2011).

5. CONCLUSION

The comprehensive histological characterization of T. somphongsi, a critically endangered cyprinid endemic to Thailand's central watersheds, provides novel insights into its tissue‐level organization while establishing crucial baseline data for conservation strategies. Our findings reveal sophisticated adaptations in digestive, reproductive and respiratory systems that reflect the species’ evolutionary response to its highly restricted habitat requirements. The asynchronous reproductive pattern, specialized gill architecture and distinctive intestinal modifications demonstrate unique physiological adaptations that inform habitat requirements and management priorities, particularly critical given the species’ extremely limited distribution and vulnerability to extinction. The documentation of sexual maturity at 2.2 cm total length, combined with detailed tissue‐level parameters, provides scientific metrics for population monitoring and protection measures crucial for preserving one of the world's 100 most threatened species. These findings bridge critical knowledge gaps in cyprinid biology while establishing an evidence‐based framework for conservation planning. Future investigations should focus on temporal variations in tissue organization and physiological responses to environmental stressors, particularly relevant given the increasing anthropogenic pressures on remaining populations. This integrated understanding of biology is essential for developing effective conservation strategies that ensure the long‐term survival of this critically endangered endemic species in its increasingly threatened natural habitat.

AUTHOR CONTRIBUTIONS

Tappadit Mitparian: Literature reviews; data analysis; methodology and writing of the original manuscript. Sitthi Kulabtong, and Chavalit Vidthayanon: Sample collection and editing manuscript. Narit Thaochan, Atsuo Iida, Koraon Wongkamhaeng, Korakot Nganvongpanit, Anan Kenthao: Data analysis and editing manuscript. Sinlapachai Senarat: Conceptualization; data generation; methodology; manuscript preparation; editing manuscript and supervision. All authors discussed the results and contributed to the final manuscript.

CONFLICT OF INTEREST STATEMENT

We have no conflict of interest to declare.

ACKNOWLEDGMENTS

This work was supported by the Microtechnique Laboratory, Division of Biological Science, Faculty of Science, Prince of Songkla University, Songkhla, supporting the chemical reagents and facilities. This research was also partially supported by Chiang Mai University.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available upon request from the corresponding author.

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Data Availability Statement

The data that support the findings of this study are available upon request from the corresponding author.

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