Macro and microscopical observations of a blue shark, Prionace glauca (Elasmobranchii: Carcharhiniformes), diprosopus embryo from South Atlantic OceanAbstract
A case of diprosopia anomaly in a blue shark (Prionace glauca) embryo from the South Atlantic Ocean is described. A detailed morphological description of internal organs was provided. The sample came from a pregnant female caught near Trindade Island (19°29'30”S, 028°20’00”W) in Southeast Brazil, with three normal embryos and one with external morphological abnormalities. The anomalous embryo was a male, with deformities in the skull and curvature of the spinal column twisted from the end of the first dorsal fin to the end of the caudal fin in a counterclockwise direction. Furthermore, two faces were observed, the right one with few alterations, while the left one was fused to it, with deformations in the eyes, mouth and nostril. The stomach was the only organ to show alteration in its shape, macroscopically. Organs of the digestive tract, renal structures and rectal gland was analyzed microscopically, and no alteration were observed in the samples. This is the first description of the internal organ of a specimen with diprosopia. These results suggest that, in addition to other factors, the survival of the embryo could be related to their ability to feed and escape predators in case to survive birth.
Keywords:
anomaly; morphology; reproduction; placental viviparity; Southeast Brazil
Resumo
Um caso de anomalia do tipo diprosopia em um embrião de tubarão azul (Prionace glauca) capturado no Oceano Atlântico Sul é descrito. Uma descrição morfológica detalhada foi feita dos órgãos internos. A amostra foi oriunda de uma fêmea grávida, capturada próxima à Ilha Trindade (19°29'30”S, 028°20’00”W) no Brasil, com três embriões normais e um com anomalias morfológicas externas. O embrião anômalo era macho, com deformidades no crânio e curvatura da coluna vertebral que estava torcida da extremidade da primeira nadadeira dorsal até a extremidade da nadadeira caudal no sentido anti-horário. Além disso, foram observadas duas faces, a direita com poucas deformidades e a esquerda fusionada a ela, com deformações nos olhos, boca e narina. O estômago foi o único órgão que apresentou variação morfológica macroscopicamente. Os órgãos do trato digestivo, estrutura dos rins e a glândula retal foram analisadas microscopicamente e nenhuma alteração foi encontrada nas amostras. Este é a primeira descrição dos órgãos internos de um espécime com diprosopia. Estes resultados sugerem, além de outros fatores, que a sobrevivência do embrião pode estar com a capacidade de se alimentar e escapar de predadores em caso de nascimento.
Palavras-chave:
anomalia; morfologia; reprodução; viviparidade placentária; sudeste do Brasil
1. Introduction
Developmental anomaly, or embryonic anomalies, is a term for a condition that is present at parturition or hatching and results from an abnormal or irregular alteration during the developmental process (Luer and Wyffels, 2022). These anomalies are often associated with the cephalic region, including dicephaly, diprosopia, cyclopia, and duplicate or absent components of the face (Santos and Gadig, 2014; Goto et al., 1981; Guida et al., 2014; Muñoz‐Osorio et al., 2013; Ramírez-Amaro et al., 2019; Sans-Coma et al., 2017). The blue shark (Prionace glauca Linnaeus, 1758) has a higher number of bicephalism and diprosopia reported in comparison to other elasmobranch species (Ramírez-Amaro et al., 2019). Cases of embryonic malformations in blue sharks have been reported in Brazil, mainly in the southern and southeast regions, associated with trunk and cephalic region abnormalities like diprosopia, cyclopia, and mouth malformation (Cabanillas-Torpoco et al., 2023; Galván-Magaña et al., 2011; Lamarca et al., 2017; Mancini et al., 2006).
The blue shark, a significant fishery resource, is caught by various fishing methods, including longline, seine net, and gill net fisheries, in both large-scale and small-scale fleets as a target species or as bycatch (Barreto et al., 2016; Montealegre-Quijano and Vooren, 2010). It is valued not only for its fins but also for its meat. This pelagic species, reaching up to 380 cm in total length, exhibits the highest growth rates among pelagic sharks and is widely distributed in tropical and temperate waters globally (Silva et al., 2021). The species uses a placental viviparous strategy, with a gestation period of 9-12 months (Balon, 1975; Bodas and Ferreira, 2009; Compagno and Niem, 1998; Dulvy and Reynolds, 1997; Legat and Vooren, 2008) and higher fecundity than other species (4-63 individuals, maximum 135 embryos) (Compagno and Niem, 1998; Rigby et al., 2019).
Among all the recorded elasmobranch species abnormalities, there are very few records of internal organ deformities or morphological descriptions (Luer and Wyffels, 2022; Sans-Coma et al., 2017). Despite the external anomaly description providing insights into the specimen's survival, it is essential to understand whether the anomaly has any consequences for the organ development. This study is the first to offer a morphological description of internal organs in a P. glauca diprosopus embryo with a spiral twisted spine in the South Atlantic Ocean, in the state of Espírito Santo, Brazil.
2. Materials and Methods
Care and use regulations for experimental animal welfare were not applicable in this study due to the nature of data collection from commercial fishing landings, which, in this case, does not require a license from the Ethics Committee on the Use of Animals, given the specimens are deceased bodies. A female blue shark (Prionace glauca) was caught northeast of Trindade Island, Espírito Santo state, under coordinates 19°29’30”S, 028°20’00”W. The specimens were captured using a surface longline with a length of 40 nautical miles and one thousand size 16 circle hooks on a 16-meter vessel that departed on February 15th and returned on March 7th, 2021.
During the fishery, a pregnant female weighing 40 kg (~ 218.00 cm TL) with four embryos (three normal and one abnormal, as reported by the fishers) was caught, and the normal embryos were released; furthermore, there is no information about how many or if abortion may have happened during capture. The abnormal embryo (Figure 1) was donated by the fisherman. Initially preserved in 70% alcohol, it was later deposited at the Laboratório de Dinâmica de Populações Marinhas at the Instituto Federal de Ciência e Tecnologia do Espírito Santo (Piúma campus), Brazil, with the catalog number: PGL 001. The malformed embryo underwent radiographic examination on left and right dorsoventral, ventrodorsal, and laterolateral views to better visualize the anatomy and facilitate morphological and abnormality diagnosis.
Prionace glauca abnormal embryo. (A) Dorsal view showing one head, two faces (F1 and F2) and two snouts, a single trunk, pectoral and dorsal fins and the clasper; (B) Right ventral view of the face 1 (F1) one with a lateral mouth (Mr) and an incomplete labial sorrow; (C) Lateral view of the face 1 and the lordosis (LO) in the spine; (D) Left ventral view showing the second malformed face (F2) with no formed nostrils, a small fissure in the portion of the mouth (Ml) and the eyes in the center of the ventral face; (E) Right ventral lateral view X-ray plate showing the gills (G) and the pectoral fin (Pf), the spine twisted (St) with torsion starting at the end of the first dorsal fin and extending until de caudal fin in a counterclockwise direction and (F) left lateral x-ray plate from the view of the face 2 showing the lordosis process. Scale bar 4 cm.
Following radiography, a morphometric assessment (in mm) was conducted on the embryo, recording its weight (g) and sex. External abnormalities were described, and the embryo's internal anatomy was analyzed. Embryonic development followed Bruno (2016). Morphometric data for blue shark embryos caught in Southeast and South Brazil (Caltabellotta, 2009) were used to estimate the embryo's total length in the absence of abnormality. A growth curve for P. glauca embryos (Caltabellotta, 2009) was used to estimate the embryo's gestational age.
2.1. Histological analysis
After the dissection, cross sections (3-5 mm) of the esophagus, stomach, intestine, rectal gland, and kidney were cut into anterior, middle, and posterior regions. Tissues were fixed in 10% formalin for 24 hours and stored in 70% ethanol until processing. Routine paraffin embedding was followed, sections were cut at 5 μm, deparaffinized, and rehydrated stepwise through an ethanol series. Subsequently, tissues were processed for routine Hematoxylin-eosin (HE) and Periodic acid-Schiff (PAS)-Alcian Blue (AB) staining, and images were captured using a stereo and light microscope with a coupled HD camera and Sigma Scan Pro 5.0 Image Software.
3. Results
3.1. External morphology
The abnormal embryo was male, displaying a single body. On the dorsal side, it featured only one first and second dorsal fin, and one caudal fin, while the ventral side showcased a pair of pectoral and pelvic fins, along with one caudal fin (Figure 1A). The embryo exhibited a single trunk with partial craniofacial duplication, fused at the fifth gill opening (Figure 1A). On the right face (F1, Figure 1), a laterally positioned mouth with an incomplete labial sorrow and well-developed jaws was observed (Figure 1B, C). The face on the left (F2, Figure 1D) lacked nostrils, the mouth appeared as a small fissure on the ventral side, and the eyes, one nearly entirely covered by dermal denticles, were joined at the center of the ventral cranium (Figure 1D). The specimen's spine exhibited torsion starting at the end of the first dorsal fin and extending counterclockwise to the caudal fin (Figure 1A, 1E, 1F). The abnormality, depicted in Figure 1A-F, was most evident in the embryo's right dorsoventral view radiograph (Figure 1E, F), revealing a hump at the end of its cranium, extending to the anterior margin of pectoral fins. Despite duplicated facial structures, the spine and internal organs were not duplicated. The embryo's weight was 79.656 g, with the liver at 3.172 g, stomach at 2.626 g, and intestine at 1.232 g.
The embryo, in phase III of development, exhibited blue pigmentation covering the dorsal body, pectoral, and caudal fins, while the ventral region lacked pigmentation. Detailed abnormalities with morphometric assessment revealed differences in mouth size, internarial space, and eye length (Table 1). The total length of 255.5 mm, estimated from the right face to the caudal fin, corresponded to an embryo with a gestational age of 6.16 months.
Morphometric measurements (mm) of blue shark (Prionace glauca) abnormal embryo caught off Espírito Santo, Brazil.
3.2. Gross morphology
The digestive tract analysis of P. glauca embryos included the orobranchial cavity, esophagus, anterior portion of the “cardiac” stomach, pyloric stomach, spiral intestine, and posterior intestine. The orobranchial zone of the face on the right featured a small oral cavity and pharynx with only one side of the lateral gills (five). Triangular teeth with high recurved cusps formed 5-10 successive tooth-seen bands in both upper and lower jaws. The left side exhibited a malformation of the oral cavity, twice as small as the other side, with no evident teeth and a separate pharynx connected to the lateral gills (five). A shared short, straight, and tubular esophagus with transverse folds at its junction with the stomach was observed. The embryo had a small two-lobed liver (Figure 2A). The twisted spine caused a torsion, changing the stomach's shape and location to the right side of the abdominal cavity (Figure 2B). The rounded stomach lacked external divisions (Figure 2B), and the intestine, larger than the stomach, appeared as a straight tube with a cylindrical valve (Figure 2C).
Diprosopus embryo of Prionace glauca with the view of interne organs. (A) The bilobuleted liver and (B). The gastrointestinal tract showing the esophagus (ES), the stomach (St), stomach pyloric (Py), anterior intestine (AI), spiral intestine (SI) and posterior intestine (PI); (C) The view of the trunk spine in the abdominal cavity. Detail of the elongated lobulated kidney (K) is visible and parallel to them the tiny epigonal organ (EO).
The rectal gland, suspended in the mesentery on the right side of the dorsal portion of the post-valvular intestine, had a small, compact shape with visible lobulations, measuring 13 mm in total length (5% of the embryo's total length).
The kidney, paired and elongated, extended from the anterior to the caudal part of the abdominal cavity (left 2.8mm and right 3.1mm), dorsoventrally flattened, with visible lobulations (Figure 2C). The epigonal organ appeared parallel to the kidney as a tiny elongated tube (Figure 2C).
3.3. Histological observations
3.1.1. Esophagus
The esophagus exhibited a short length connecting the pharynx and the stomach, lacking esophageal sphincters. The esophageal wall, distinct from the stomach, comprised mucosa, submucosa, muscular, and serosa layers (Figure 3A). The mucosal layer featured longitudinal folds, cone-shaped and branched papillae with desquamated stratified squamous epithelium, showing acid and neutral mucin-like cells via PAS-AB staining (Figure 3B-D). The submucosa contained loose connective tissue with longitudinal and transverse collagen fibers and numerous blood vessels (Figure 3C). A muscularis mucosa was absent, while the organ of Leydig, a lymphomyeloid tissue mass, was dispersed in the submucosa (Figure 3C, 3E) and composed of blast cells, granulocytes (macrophages), and lymphocytes (leucocytes) (Figure 3E). The muscular layer displayed thick circular, longitudinally striated muscles along the organ (Figure 3C), and externally, there was a serosa with a subserosal nerve plexus.
Cross-section of the esophagus. (A) The esophageal wall constituted by mucosa (M), submucosa (Sub), muscular (Mu), serosa (Se) and nervous plexus (
). (Hematoxylin-Eosin, scale bar = 2mm); (B) The thick circular longitudinal striated muscles of the muscularis run along the organ (Masson trichrome, scale bar = 500 µm); (C) The mucosa was formed by longitudinal folds (LF), cone-shaped and branched easily desquamated (deficient fixation process) stratified squamous epithelium (DSE) (Hematoxylin-Eosin, scale bar = 500 µm); (D) with acid and neutral mucin-like cells (PAS-AB, scale bar = 50 µm); (E) The Leydig organ (OL) in the superficial portion of the submucosa (Sub) is visible and the mass of limphomyeloid tissue (HE, scale bar = 50 µm).
3.1.2. Stomach
The stomach exhibited four layers: mucosa, submucosa, muscular, and serosa (Figure 4A). Long branched folds decreased in height from the cardiac to pyloric portions, transitioning with changes in epithelial and glandular characteristics. In the anterior cardiac portion, a gradual shift from esophageal stratified epithelium to columnar tall mucous cells with oval nuclei in the basal third of the cell lined the stomach surface was evident (Figure 4A, B). Neutral PAS-AB reactive mucus, microvilli, and an absence of gastric pits characterized this region (Figure 4C). The epithelial folds distinguish the anterior from the middle portion that showed gastric pits, gland formation, and abundant blood vessels in the lamina propria and submucosa (Figure 5A, B). Muscular layers had bundles arranged circularly and longitudinally with a nerve plexus between them (Figure 5B). The epithelial cells proliferate and penetrate the lamina propria and submucosa to form gastric glands (Figure 5C, D). In this stage of embryonic development, short non-ramifying tubules were seen. These glands open into the lumen and are composed of only one type of secretory cell with microvilli on their apical surface (Figure 5D). The gland's epithelium varied from tall cubic cells near the mucosa with oval nuclei (Figure 5C) to short cubic cells in the upper portion (Figure 5D). Deep into the submucosa, the gland had a cell composition similar to oxynticopeptic or oxyntopeptic cells and showed no positive reaction to PAS-AB staining (Figure 5D). In the posterior/pyloric portion of the stomach, the mucosa underwent a change marked by the sudden disappearance of gastric pit glands (Figure 6). Gastric pits became narrow and short, and started to extend throughout the mucosal thickness (Figure 6). The epithelium remained similar to that of the anterior portion. The submucosa became more prominent, filling the space among the pits. A cluster of cells resembling a lymph node was observed close to the surface epithelium (Figure 6), along with lymph vessels in the submucosa (Figure 6).
(A) Cross-section of the anterior portion of the stomach of an abnormal embryo of Prionace glauca, revealing the branched long folds transitioning from the esophagus, the mucosa (M), submucosa (Sub) and muscular (Mu). (HE, scale bar 0.5 mm); (B) in the mucosa, a line of microvilli is seen in the surface of columnar tall mucous cells (
); (C) The neutral reactive cells by PAS-AB (scale bar = 50 µm).
(A) Cross-section of the medium portion of the stomach (equivalent to descending cardiac), the mucosa (M), submucosa (Sub), muscular (Mu) and serosa (Se). The transition between the gastric and pyloric portions showing the morphological variation of the gastric pits (arrow) (HE, scale bar = 2 mm); (B) The development of the gastric pits (GP) and gastric glands (GG) in the mucosa which will penetrate the submucosa (Sub), blood vessels (BV) are abundant in this region and between two layers of smooth muscle (Mu) the nerve plexus (PN) is seen and close to them the serosa (Se) (HE, scale bar = 200 µm); (C) The gastric glands (GG) are non-ramified and secretory cell with microvilli in the apical surface forming the gastric pits (GP); (D) These cells varying from tall cubic cells in the upper portion to short cells distributed deep into the submucosa (Sub), these cells are similar to that oxynticopeptic cells (PAS-AB, scale bar = 50 µm).
Cross-section of the transition between the medium (MP) and posterior region of the stomach. In the posterior portion (PP), the gastric glands are absent, and the epithelium is connected with the submucosa (Sub) directly. Gastric pits (GP) are narrow and short. A lymph node-like cluster of cells (LN) is seen close to the surface epithelium and lymph vessel (LN) in the submucosa (Sub). (Hematoxylin-Eosin, scale bar = 500 µm).
3.1.3. Intestine
Due to material limitations, we could only analyze the spiral valve of the intestine. The mucosa displayed numerous folds (Figure 7), with funnel-shaped epithelium consisting of spaced folds lined by a single layer of simple columnar cells with basal nuclei (Figure 7). The submucosa was composed of loose connective tissue, lacking submucosal glands. The muscular layer exhibited thin bundles.
Cross-section of the intestine showing the spiral valve (SV) present in the anterior (Hematoxylin-Eosin, scale bar = 0.5 mm) and medium portion of the intestine, the funnel-shaped folds (head arrow) of mucosa and submucosa are line by a single layer of simple columnar cells. E = epithelium; Sub = submucosa; Mu = muscle layer. (Hematoxylin-Eosin, scale bar = 0.5 mm).
3.1.4. Rectal gland
Three distinct regions of the adult gland were observed: the outer connective tissue of the capsule, secretory parenchyma, and the central collecting duct of the transitional epithelium (Figure 8A). The outer capsule, a vascularized connective tissue, featured a peripheral circumferential arterial system feeding capillaries that drained into a central venous system (Figure 8B). Smooth muscle-like tissue surrounded the organ and appeared to invaginate the parenchyma, forming lobules with a dense capillary network close to secretory tubules (Figure 8B, 8C). The tubules showed no regional specialization, and secretory cells appeared homogeneous, cuboidal with basal granular nuclei (Figure 8C). Apparently, the secretory cell morphology seemed to be the same from the periphery to the central collecting duct. Differences in tubule lumen size were noted between the outer capsule, the middle area, and those near the central collecting duct (Figure 8B). Cells near the outer rim were densely packed and had a small lumen, whereas those near the central duct were loosely bundled, forming medium and large tubules with a wide inner diameter (Figure 8C). Tubules opened into the central duct with a seemingly simple cylindrical ciliated epithelium lining the lumen. A bundle of capillaries connected the blood sinuses in the central collecting duct and the peripheral region (Figure 8D).
Cross section of the rectal gland of the abnormal embryo of Prionace glauca. (A) The capsule (CAP) surrounds the gland and below it the connective tissue layer (CT), the interlobular septum (IS) dividing the secretory parenchyma (SP), the blood drainage is performed by the main artery (ART) and vast blood vessels present in the gland. In the innermost part of the organ is seen the central lumen (CL); (B) Detail of the interlobular septum (asterisk and dot line) partially dividing the secretory parenchyma (SP) with a dense capillary network (arrowhead); (C) The secretory cells (SC, arrow) are cubic with basal nuclei and compound the secretory tubules (ST) which show different lumen (L) dimeter. Blood vessels are abundant, and erythrocytes are easily seen in this tissue; (D) The central lumen (CL) with microvilli in the epithelium surface (arrowhead), the venous sinuses (VS) with a massive number of erythrocytes (E). (A) Masson trichrome (scale bar = 500 µm); (B) He (scale bar = 200 µm); (C) and (D) (Hematoxylin-Eosin, scale bar = 50 µm).
3.1.5. Kidney
The renal structure in this diprosopus individual showed no alterations. The kidney featured two zones from the lateral to the medial part of the organ. The thin peripheral bundle zone with packed tubules and glomeruli (Figure 9A, 9B). The urinary pole was directed toward this zone (Figure 9A). The central larger sinus zone formed the major mass of the renal parenchyma (Figure 9A). Sinus blood with numerous large capillaries was prominent, especially at the border of the bundle zone extending into the interbundle area in the sinus zone (Figure 9A, 9C). Small interconnected blood sinuses surround the tubules in this area (Figure 9B). Viewing by transverse cut, the renal corpuscles varied in shape from ovoid to elongated, distributed linearly in groups within the parenchyma (Figure 9B), and visually variable in size.
The kidney configuration of the abnormal embryo of Prionace glauca. (A) Two distinct zones (dotted line) are seen forming the lateral (bundle zone - BZ) and the central portion (sinus zone - SZ) separated by the line of renal corpuscles (RC). The urinary poles (UP) of the renal corpuscles are points towards the bundle zone where is seen the collecting duct (CD) (HE, scale bar = 500 µm); (B) Detail of the bundle zone with the line of renal corpuscles which is compound by the glomeruli, Bowman´s capsule and distal segment (D) and above of the RC the colleting duct is seen in the cortical area. In the sinus zone a vast of capillaries (C) is seen; (C) Detail of the sinus zone showing capillaries widely in size and glomerulus (G) in different sizes in both sinus and bundle zones. (Hematoxylin-Eosin, scale bar = 200 µm).
4. Discussion
Embryonic anomalies are widespread among elasmobranchs globally, particularly in viviparous species, a fact that could be associated with the continuous maternal nutritional supply during the gestation ensuring the development of anomalous embryos (Galvan-Magaña et al., 2011). The first documented case of morphological abnormalities in Prionace glauca embryos dates back to 1963 in the Mediterranean Sea, involving two heads and a deformed spine (Parenzan, 1979). Since then, diverse abnormalities such as diprosopia, twisted spine, deformed snout, cyclopia, deformed rostrum, and mouth have been reported globally for blue shark embryos (Cabanillas-Torpoco et al., 2023; Ehemann et al., 2016; Galván-Maganã et al., 2011; Pastén-Marambio et al., 2018; Ramiréz-Amaro et al., 2019; Rodriguez-Romero et al., 2019; present study). However, previous studies lacked microscopic assessments of internal organs, making this work the first to comprehensively analyze the anomaly, providing microscopic morphological characteristics of the digestive tract, kidney, and rectal gland, in addition to the conventional macroscopic description of external and internal morphological anomalies.
Among the reported abnormalities in blue shark embryos, bicephaly and diprosopus are the most common. Bicephaly involves conjoined twins with two separate heads on one trunk, sometimes with duplicated internal organs (Sans-Coma et al., 2017; Witschi, 1952). This anomaly is frequently observed in various shark species, such as Galeorhinus galeus, Carcharhinus leucas, Rhizoprionodon porosus, Mustelus higmani, Galeus atlanticus and Rhizoprionodon lalandii (Delpiani et al., 2011; Santos and Gadig, 2014; Ehemann et al., 2016; Prado et al., 2020; Sans-Coma et al., 2017; Wagner et al., 2013), including P. glauca (Bejarano-Álvarez et al., 2011; Ehemann et al., 2016; Galván-Magaña et al., 2011; Goto et al., 1981; Ramírez-Amaro et al., 2019). Diprosopus, often confused with bicephaly, is characterized by partial or complete duplication of the face, forebrain, and upper vertebral column (Biasibetti et al., 2011; Spencer, 2000). Both conditions arise from nearly parallel notochords on an embryonic disc, close together caudally, with varying degrees of separation rostrally (Spencer, 2000).
The embryo in this study exhibited parapagus diprosopia, with two faces fused laterally, one fully formed (right face) and the other (left face) displaying a rostrum with rudimentary cranial structures (Biasibetti et al., 2011; Spencer, 2000). The right face was larger, with an asymmetrical mouth and jaw, while the left face had a small fissure and no ventral jaw. It is important to note that, unlike most previous studies on P. glauca embryos, we did not consider the embryo to have two distinct heads. Furthermore, various malformations have been recorded for blue shark embryos in southern Brazil, the west coast of Baja California Sur, and the Peruvian coast (Kanagusuku et al., 2020; Mancini et al., 2006; Rodriguez-Romero et al., 2019), associated with factors such as malnutrition, zygotic division, genetic abnormalities, parasites, and pollution (Heupel et al., 1999; Kanagusuku et al., 2020; Mancini et al., 2006; Rodriguez-Romero et al., 2019). These findings highlight the multifactorial nature of malformations in different morphological structures, underscoring the importance of continued research in understanding these anomalies and their potential causes.
Embryonic development, despite the observed abnormalities, exhibits no delay in general external morphology consistent with typical embryonic stages observed in blue sharks. The estimated size at birth for this species ranges from approximately 350-600 mm in total length after a gestation period of 9-12 months (Pratt, 1979). In Brazil, births are anticipated between September and December (Amorim et al., 2020; Montealegre-Quijano et al., 2014). Considering the estimated total length (~255 mm), the embryo was in the last phase (V), adopting the classification by Legat and Vooren (2008) and the phase III from IV by Bruno (2016), which means in both cases the embryo was in the late phase of development. The gestation period of the abnormal embryo analyzed here, the reduced size compared to the estimated birth size for the species may be attributed to factors such as a small uterus with a large number of embryos, potentially causing issues in embryonic development, as observed by Galván-Magaña et al. (2011) for an abnormal blue shark embryo in the Mexican Pacific Ocean. The intrauterine pressure may induce lateral and posterior curvature of the spine, resulting in a screw-shaped trunk (Pastore and Prato, 1989), as observed in this study. Other factors contributing to reduced size include low nutrient transfer, similar to observations in a P. glauca bicephaly (Galván-Magaña et al., 2011), along with potential factors like parasitic infections, injuries, vitamin C deficiency, or congenital diseases (Heupel et al., 1999).
The internal organs of the abnormal embryo analyzed, were fully formed, with macroscopic morphology changes observed in the stomach, displaying a rounded shape differing from the typical J-shaped form observed in sharks (Holmgren and Nilsson, 1999). This alteration, not previously reported in blue sharks, is hypothesized to result from the screw-shaped trunk with counterclockwise torsion affecting the organ on the opposite side (left). Twisted torsion is frequently recorded in malformed P. glauca embryos (Mancini et al., 2006; Kanagusuku et al., 2020; Ramírez-Amaro et al., 2019) and in other Carcharhinidae species such as Rhizoprionodon lalandii (Prado et al., 2020). Spinal deformities in sharks have been associated with scoliosis, central fusion, and arthritic changes (Bonfil, 1989), particularly in P. glauca, where multiple abnormalities like diprosopia and scoliokyphosis have been reported (Ramírez-Amaro et al., 2019). Furthermore, the liver was smaller than observed for embryos at the same length observed by Melo (2018), probably associated with the facts mentioned before. Changes in internal organs have also been documented in this species, such as a face-duplicated embryo from the Pacific Ocean, which exhibited four livers with one atrophied, while other internal organs remained normal (Rodriguez-Romero et al., 2019).
The two faces shared a unique esophagus, which exhibited no change in microscopic composition. Despite the desquamated epithelial surface of the organ, the stratified squamous epithelium without cilia resembles that observed in the white-spotted bamboo Chiloscyllium plagiosum and the multilayered squamous epithelium of most teleost fishes (Harder, 1975). It differs from the adult dogfish Squalus acanthias, which possesses cilia (Leake, 1975). Changes in esophageal composition may relate to feeding adaptation, facilitating the ingestion of various food sizes (Shalaby, 2020). In this study, the presence of cells with neutral mucus secretion, resembling goblet cells on the esophageal surface (see Figure 3D), may aid in mucus secretion for food transport and epithelial protection against proteolytic degradation caused by stomach acid when the stomach is fully differentiated and active. Similar observations were made for normal embryos 35 days after hatching of the white-spotted bamboo, including the presence of tall columnar epithelial cells associated with goblet cells on the esophageal surface, suggesting the same role in food transportation and mucosal protection (Xu et al., 2015). Gastric glands in the blue shark embryo were small with cubic cells, displaying no secretion activity, making it difficult to identify the type of gastric gland cell. For teleost and some elasmobranchs, only one gland cell type is recognized, the oxynticopeptic cells, which contain acidophilic granules indicating they are pepsinogen-secreting and acid-secreting cells (Holmgren and Nilsson, 1999; Wilson and Castro, 2010).
The absence of glands in the posterior region of the stomach in the analyzed embryo aligns with expectations for some elasmobranchs (Holmgren and Nilsson, 1999). The mucosa in this posterior region is narrow and short with developing pits and an extensive submucosa. It is likely that the appearance of the epithelium will change as the embryo approaches birth and starts feeding on dry food, given that the stimulus for digesting food is the expansion of the stomach wall (Holmgren and Nilsson, 1999), evident from the presence of the extensive submucosa. Various factors, including long gastric emptying times (Holmgren and Nilsson, 1999), high mucous content in the epithelium, and the extended submucosa, could contribute to the absence of gastric glands in this region. These elements might play a protective role against perforation by prey exoskeletons, considering these animals likely store food (Chatchavalvanich et al., 2006).
The intestine of the abnormal blue shark embryo is short, exhibiting spiral folds formed from the infolding of the intestinal mucosa and submucosa, a common feature in elasmobranch species. The spiral valve enhances surface area for absorption and extends digesta transit time, ultimately reducing the size of the intestinal area (Bucking, 2015; Holmgren and Nilsson, 1999). The number of turns in the spiral valve is linked to diet, allowing surface absorption and delaying digestion (Holmgren and Nilsson, 1999); the abnormal embryo analyzed here demonstrates normal anatomy of the spiral intestine. As a placental viviparous shark, nutrients and macromolecules are actively transported to the embryo by the placenta (Otake and Mizue, 1986). This likely does not interfere with nutritional feeding during embryonic phases of abnormal embryos, as no compromised fetal development or alterations in internal organ composition were found.
The earliest primordia of the rectal gland in elasmobranch embryos appear as protrusions of the gut, typically at the junction of the spiral valve and rectum (Fishelson et al., 2004). For species like Lago omanensis and Squalus acanthias, it appears in embryos with total lengths of 14 mm and 15 mm, respectively, (Fishelson et al., 2004; Hoskins, 1917), and for the latter species, the gland characteristics of an adult emerge when the size of 200 mm is reached (Hoskins, 1917). There is no description of this organ for adults or even embryos of blue sharks. In the blue shark abnormal embryo analyzed here, the rectal gland corresponds to 5% of the body length (255.5 mm, with a rectal gland length of 13 mm), which differs from other small shark species. The distinct lobulated rectal gland observed is similar to Carcharhinus leucas adults (Pillans et al., 2008) but differs from the digitiform glands observed by Larsen et al. (2019) in other Carcharhinidae species. The same author indicated that the rectal gland's anatomy is suggested to be influenced by taxonomic filiation rather than depth, opposing the observations in the present study. Further studies are required to test this prediction, as this is the first description of this structure in a blue shark species.
The secretory cells of the rectal gland analyzed in the present study showed no distinct areas (small and large cells), and some secretory tubules branched, displaying a large lumen diameter in the middle portion of the organ. The large lumen is commonly observed in elasmobranchs (see Pillans et al., 2008 and Melo et al., 2021) and is suggested to increase the tubule’s surface area for secreting salt (Newbound, O’Shea, 2001). Since the abnormal embryo could be near term, this association function may indicate a possible activation of salt transport near birth.
The kidney, gills, and rectal gland play significant roles in marine and euryhaline elasmobranch osmoregulation, and kidneys are the primary site where plasma urea regulation takes place (Lacy and Reale, 1999). In the uterus environment, urea is present for the entire gestation period in viviparous species. A possible inability of the embryo to regulate urea, consequently leading to osmotic pressure, is suggested during early development (Prince Junior and Daiber, 1967). For viviparous species like M. canis, the kidney becomes functional in the late stage of embryonic development (Prince Junior and Daiber, 1967). Following this pattern, the kidney of the abnormal embryo analyzed in the present study showed structures similar to those found in adult elasmobranch kidneys (see Lacy and Reale, 1985). Regardless of the embryonic malformation, development was not affected.
Great structural variations are present in the kidneys of elasmobranchs, depending on the species, sex, age, as well as individual variation (Lacy and Reale, 1985). The authors Lacy and Reale (1985) also noted conflicting observations in different studies concerning the position of the urinary pole, glomeruli, and tubule bundle in the kidney. Many differences are observed between shark and ray species. For example, in the present study, the distribution of renal corpuscles is in linear groups, adjacent to the bundle zone in the peripheral zone (or dorsal), extending to the central (or ventral) zone. Together with the tubule bundle, the urinary pole is pointed to the central zone of the kidney. Similar findings were recorded for the spiny dogfish shark Squalus acanthias (Kempton, 1953; Lacy and Reale, 1985). Although for skates like Raja erinacea, the same structures are confined to the periphery/dorsal region of the kidney (Lacy and Reale, 1985). In general, the distribution of tubule segments is comparable to the structure of the kidney of adult sharks, such as Scyliorhinus stellaris (Borghese, 1966) and Squalus acanthias (Lacy and Reale, 1985).
The anatomical variation of the renal corpuscle is a large topic of discussion, and its size in marine and freshwater elasmobranch fishes is extremely variable (Lacy and Reale, 1999). Embryonic observations of renal corpuscle development suggest that large corpuscles usually located in the central zone are older than the small ones located in the peripheral zone, where a germinal zone with the forming area of new glomeruli and renal tubules is observed (Elger et al., 2003; Kozlik’s, 1939). Differences in renal corpuscle sizes were observed in the present study, and their distribution is similar to that observed by Elger et al. (2003).
The abnormal embryo of the blue shark exhibited no discernible alterations in the internal organs under scrutiny in this study. However, the diprosopus condition seemed to manifest primarily in the external macroscopic development. Renowned for its high fecundity among sharks, the uterine area in blue sharks is purported to be relatively small, potentially influencing prenatal development (Galván-Magaña et al., 2011). These anomalies may be correlated with the species elevated embryo production (n = 135) (Compagno, 1984; Mancini et al., 2006). Notably, being a placental viviparous species, continuous nutrient supply from the mother during gestation allows the development of abnormal embryos. Galván-Magaña et al. (2011) suggested that nutritional variations could lead to distinct growth patterns in anomalous embryos, particularly in bicephalic sharks. Malformations linked to incomplete development, such as internal organ deficiencies, incomplete external eyes and snout, anophthalmia, and incomplete gill slit development (Bejarano-Alvarez et al., 2011; Galván-Magaña et al., 2011; Rodriguez-Romero et al., 2019), may be associated with inadequate nutritional input from the mother (Rodriguez-Romero et al., 2019). On the other hand, certain skeletal deformities may be suggested as a genetic anomaly inherent in elasmobranchs (Heupel et al., 1999).
The underlying causes of such malformations remain unknown to the scientific community. Our data show that diprosopus abnormalities in this species does not interfere in the development of the main digestive organs for this blue shark embryo. Potential correlations between biological and environmental factors might influence embryonic development. It is well known that factors such as elevated heavy metal content and adverse environmental conditions have been linked to embryonic malformations in various fish species (Heupel et al., 1999; Kanagusuku et al., 2020, Wagner et al., 2013; Zaera and Johnsen, 2011). Unfortunately, heavy metal analysis was precluded in the material under this study due to specimen storage in alcohol on the vessel. Additionally, the anomaly may be a congenital change with a low probability of occurrence, but given the blue shark's status as one of the most abundant shark species globally, such cases are recurrent.
Acknowledgements
Instituto Federal de Educação, Ciência e Tecnologia do Espírito Santo (IFES) for the financial support (PRODIF PRPPG 08/2024). We extend our gratitude to Roberta Cardozo de Paiva Garcia and Bruno Muniz Vieira for facilitating the donation of the abnormal embryo and sharing catch data. Special thanks to Focus Diagnóstico Veterinário and veterinarian Juliana Guadalupe Souza Belmondes for conducting radiographies on the embryo.
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