Molecular identification, early development and distribution of larval blunthead puffer Sphoeroides pachygaster (Teleostei: Tetraodontidae) off southeastern Brazil

Henrique Grande¹ , Ana C. T. Bonecker², Mariana M. Julio², Mario Katsuragawa¹, Maria de Lourdes Zani-Teixeira¹, Mateus G. Chuqui¹, Artur R. Spirgatis¹, Gabriela B. França¹, Frederico P. Brandini¹ and Cláudia A. P. Namiki¹

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Abstract​

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Introduction​

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The Tetraodontidae is the most diverse family within the Tetraodontiformes, encompassing 28 genera and about 193 valid species (Fricke et al., 2025). These predominantly tropical and subtropical fishes inhabit a wide range of environments, including open waters, reefs (Alfaro et al., 2007; Yamanoue et al., 2011), shallow coastal waters (Froese, Pauly, 2024), and even brackish or freshwater habitats (Dekkers, 1975; Ebert, 2001; Yamanoue et al., 2008).

Species of the Tetraodontidae have a varied diet, including carnivorous, omnivorous, herbivorous, and corallivorous habits (Stump et al., 2018). They play crucial ecological roles, regulating benthic community structures and serving as key links in the food chain (Eduardo et al., 2020). Information on the reproductive biology of Sphoeroides pachygaster (Müller & Troschel, 1848) is limited to brief studies in the Mediterranean Sea, which suggest a prolonged reproductive period with advanced ovaries observed in winter and late summer (Ragonese et al., 2001). They are characterized by their oval bodies and ability to inflate by ingesting water or air (Wainwright, Turingan, 1997). Typically, these fishes are small to medium, with well-developed heads and beak-like modified jaws formed by fused teeth, resulting in two upper and two lower plates. Additionally, they lack pelvic fins as well as dorsal and anal spines (Cervigón, 1995).

In Brazil, fourteen valid species of the Tetraodontidae are reported in five genera: Arothron Müller, 1841, Canthigaster Swainson, 1839, Colomesus Gill, 1884, Lagocephalus Swainson, 1839, and Sphoeroides (Lacepede, 1798) (Menezes et al.,2003;Díaz de Astarloa et al.,2003;Pinheiro et al.,2018). The blunthead pufferfish, Sphoeroides pachygaster, typically inhabit depths greater than 100 m, being recorded in waters as deep as 480 m (Shipp, 1974; Matsuura, Tyler, 1997). Adult S. pachygaster primarily feeds on squids, cuttlefish, octopuses, and small bony fishes (Smith, Heemstra, 1986; Psomadakis et al., 2008). The larvae and juveniles of S. pachygaster are pelagic, while adults prefer benthic habitats such as sandy, muddy, and rocky bottoms (Robins, Ray, 1986; Tortonese, 1986).

Sphoeroides pachygaster has a circumglobal distribution in tropical and temperate seas (Carpenter, De Angelis, 2016) and is widely found in the western Atlantic, from New Jersey (USA) to Argentina (Cousseau, Batista, 1976; Cervigón, 1995; Smith, 1997; Figueiredo, Menezes, 2000; Pinheiro et al., 2018). Phylogenetic studies suggest that S. pachygaster is basal within the genus Sphoeroides, with an early divergence from other species of the genus (Amaral et al., 2013; Araújo et al., 2023; Hunt et al., 2023). Furthermore, molecular analyses and phenotypic differences at the population level (Shipp, 1974; Amaral et al., 2013; Araújo et al., 2023; Hunt et al., 2023) indicated that S. pachygaster may represent a species complex, potentially comprising several morphologically similar but as yet undescribed species currently grouped under the same name.

Although basic meristic characters of S. pachygaster larvae have been reported (Moser, 1996; Lyczkowski-Shultz, 2005; Fahay, 2007), a detailed description of their larval stages is still unavailable. Understanding the larval morphology of S. pachygaster in the western South Atlantic may help distinguish potential cryptic lineages and verify whether the larvae from the Brazilian coast belong to the same morphogenetic lineage as those from other regions. In this context, we describe the larval developmental stages of S. pachygaster, focusing on morphology, pigmentation patterns, the development of rays and fins, and confirm species identification through DNA barcode analysis. Additionally, we provide new insights into the distribution of larvae in the western South Atlantic.

Material and methods

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Molecular identification. DNA extraction, PCR, and sequencing. Due to the scarcity of diagnostic morphological characters of S. pachygaster larvae, especially during the early stages of development, we employed molecular techniques to confirm the identification of specimens in the preflexion stage. The three larvae used in this study (n = 3) had a total body length of 1.68, 1.74, and 1.97 mm. Prior to DNA extraction, we washed and hydrated the tissue with TE 1× (Tris-HCl 10 mM; EDTA 1 mM; pH 8.0) and placed each individual in 1.5 mL microtubes. Due to their small size, we used whole larvae for DNA extraction instead of tissue samples, as typically done for adult fish.

Following the manufacturer’s protocol, we extracted Genomic DNA using the DNEasy Blood & Tissue Kit (Qiagen) and quantified it with a NanoDrop Lite spectrophotometer (Thermo Scientific). We amplified a fragment of 600–700 base pairs of the Cytochrome C Oxidase I (COI) gene using Polymerase Chain Reaction (PCR) with the primers Fish F1 and Fish R1, as recommended by Ward et al. (2005). We performed PCR using Taq DNA Polymerase 2x Master Mix RED (1.5 mM MgCl2) (Ampliqon) in a final volume of 25 μL, containing 12.5 μL of the master mix, 10.5 μL of H₂O, 0.5 μL of each primer, and 1 μL of DNA. The thermal cycler program included an initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 94°C for 30 sec, annealing at 53°C for 90 sec, and extension at 72°C for 90 sec, with a final extension step at 72°C for 10 min. We analyzed the PCR products using 1.5% agarose gel electrophoresis (Invitrogen) and then prepared for sequencing using the Kit QIAquick PCR Purification to purify the samples. DNA sequencing was performed at the Instituto Oswaldo Cruz (FIOCRUZ).

After sequencing, we imported the results and analyzed their sequence quality using electropherograms. The COI gene sequences were edited with BioEdit 7.7.1 software (Hall, 1999), and used the Basic Local Alignment Search Tool (BLAST; Altschul et al., 1990) via GenBank network service (http://www.ncbi.nlm.nih.gov/) to identify homologous sequences in the nucleotide database. The sequences have been deposited in GenBank under accession numbers PP483800, PP4883801, and PP483802.

To interpret the relationships between the data, we constructed a phylogenetic tree using the Maximum Likelihood (ML) method. The phylogenetic tree was built using ClustalW v. 2.0 (Kumar et al., 2016) implemented in MEGA7.0 program (Kumar et al., 2016), with the HKY+G+I nucleotide substitution model (Hasegawa et al., 1985). Support for the clades was indicated in the tree, omitting bootstrap values below 70%. Pairwise genetic distances (Tab. S1) were estimated using MEGA7.0. The phylogenetic tree included sequences obtained in this study and those from the GenBank Database for four Tetraodontidae genera (Canthigaster, Lagocephalus, and Sphoeroides), with an outgroup comprising three sequences of Monacanthus ciliatus (Mitchill, 1818), family Monacanthidae (Tetraodontiformes). Species codes used for building the phylogenetic tree are provided in Tab. S2.

Morphological identification and descriptions. To identify the larvae based on morphology, we compiled information on the meristic characters and geographic distribution of all Sphoeroides species available in the literature. Identifications were based on characters proposed by Moser (1996), Lyczkowski-Shultz (2005), and Fahay (2007), including a stocky body shape during the preflexion stage, a smooth body without prickles, a rounded head profile, and pigmentation patterns.

The numbers of vertebrae/myomeres and the dorsal (D), anal (A), pectoral (P), and caudal (C) fin rays were counted once they were developed. These counts were made under a stereoscopic microscope. Larvae were photographed using a Leica M205C stereomicroscope. The Image J software was used for image capture and morphological measurements with an accuracy of 0.01 mm. Larvae were classified into preflexion and flexion stages based on the degree of notochord flexion (Ahlstrom et al., 1976).

Morphometric characters are as follows Moser (1996): body length (BL), distance from the tip of the snout to the posterior tip of the notochord; body depth (BD), vertical distance between body margins (excluding fins) through the anterior margin of the pectoral fin base; body width (BW), transverse distance between body margins at the pectoral fin base; pre-dorsal length (PDL), distance from the tip of the snout along the midline to a vertical line through the origin of the dorsal fin; pre-anal length (PAL), distance from the tip of the snout along the midline to a vertical line through the posterior edge of the anus; head length (HL), horizontal distance from the tip of the snout to the upper edge of the gill opening; snout length (SNL), horizontal distance from the tip of the snout to the anterior margin of the fleshy orbit; eye diameter (ED), maximum horizontal distance measured from the anterior to the posterior margin of the fleshy orbit; interocular distance (ID), horizontal distance between the outer edges of the eye orbits.

The ratios BD/BL, HL/BL, and ED/HL were used to characterize individuals based on body shape, head size, and eye size (Leis, Trnski, 1989). Body proportions (ID/HL, PDL/BL, PAL/BL, and BW/BL) were analyzed relative to BL using linear regression modeling to better understand differences in body proportions among larvae at various developmental stages.

Species distribution data. The study area encompasses the South Brazil Bight, a sedimentary area in the western South Atlantic Ocean along the southeast Brazilian continental margin. The bight spans approximately 350,000 km², bordered at the north by the Cabo Frio Arch at Cabo de São Tomé (22.1°S and 41°W), and at the south by the Florianópolis Shelf at Cabo de Santa Marta (28.55°S and 48.47°W) (Mohriak, 2003).

Previous records of larvae and adults of S. pachygaster in the study area were compiled from published literature (Sampaio et al., 2001; Figueiredo et al., 2002; Bernardes et al., 2005). For this study, larvae of S. pachygaster were collected during two oceanographic cruises of the project “Chemical and Biological Characterization of the Pelagic System of the Santos Basin” (Moreira et al., 2023), carried out from June to September 2019 and from December 2021 to March 2022, covering 60 stations in each cruise (Fig. 1).

FIGURE 1| Map of zooplankton sampling stations during oceanographic expeditions conducted from June to September 2019 and from December 2021 to March 2022 in the Santos basin, southeastern coast of Brazil. The abbreviations indicate Brazilian states: MG = Minas Gerais, ES = Espírito Santo, RJ = Rio de Janeiro, SP = São Paulo, PR = Paraná and SC = Santa Catarina.

We collected 1,037 zooplankton samples using a Multinet (Hydro-bios, Kiel) equipped with nine nets. At each oceanographic station, we conducted two oblique tows during the night (between 6 PM and 6 AM), one using a 200 µm mesh size and the other with a 500 µm mesh size. In each town, eight nets were used to sample predefined depth layers from the bottom to the surface (2,400–1,500; 1,500–1,100; 1,100–550; 550–150; 150–100; 100–50; 50–25; 25–0 m).

We preserved samples from the integrated tows in 99% ethanol for DNA barcode analysis. Samples from the stratified layers were immediately fixed post-collection in 4% formaldehyde buffered with sodium tetraborate. We deposited the larvae identified as S. pachygaster from the 500 μm mesh nets in the Biological Collection “Prof. Edmundo F. Nonato”, Oceanographic Institute, University of São Paulo (ColBIO) (IP49732, IP49733, IP49734, IP49735, IP49736, IP49737). Those from the 200 μm mesh nets were deposited in the Zooplankton and Ichthyoplankton Integrated Laboratory of the Universidade Federal do Rio de Janeiro (DZUFRJ 73816).

Results​

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Molecular identification. The topology from the Maximum Likelihood analysis of the obtained sequences and those from the GenBank Database confirms that the analyzed specimens are indeed S. pachygaster, supporting the morphological identification
(Fig. 2). The clade highlighted in blue shows that the sequences grouped with the expected species (interspecific p distance 0.000–0.039) with 100% bootstrap support.

FIGURE 2| Phylogenetic tree constructed using the Maximum Likelihood (ML) method. Sequences from this study include Sphoeroides pachygaster 22U, 26V, and 2W, with GenBank accession numbers PP483800, PP483801, and PP483802. Bootstrap values at the nodes: ML (> 70%). The red asterisk indicates voucher specimens.

The distance matrix (Tab. S1) indicates that the smallest genetic distance was obtained between the analyzed sequences and S. pachygaster sequences (0.00) available in the database, while the largest genetic distances were found between these sequences and Canthigaster figueiredoi Moura & Castro, 2002 and C. rostrata (Bloch, 1786) (0.23). The ML grouped these species distinctly, supported by high bootstrap values. Sphoeroides and Lagocephalus have a close genetic relationship (~0.2), with Sphoeroides being paraphyletic unless Lagocephalus is included.

Early development of Sphoeroides pachygaster. Preflexion stage. At the beginning of the preflexion stage (1.53–1.91 mm BL), larvae have 18 myomeres and show either no pigmentation or sparse pigmentation, with scattered pigment on the head and dorsal regions (Figs. 3A–F). Melanophores are concentrated along the midline behind the eyes and spread across the upper part of the abdominal cavity wall up to the first post-anal myomere (Figs. 3A–F). Ventral pigmentation is confined to the foregut and the terminal section of the gut (Figs. 3A–F). The pectoral fins are already visible (Figs. 3E, F), while the dorsal and anal fins have not yet formed.

FIGURE 3| Lateral, dorsal, and ventral views of Sphoeroides pachygaster larvae in the preflexion (A–L) and flexion (M–R) stages.

As preflexion larvae develop (2.90–4.16 mm BL), the pigmentation pattern on the dorsal and lateral parts of the body becomes more pronounced. From 2.90 mm BL onwards, pigment distribution along the body is visible, extending to the end of the pre-anal myomeres, with the posterior third of the larva remaining pigment-free (Figs. 3G–I). At this stage, the pectoral fins are already developed with 16 to 17 rays (Tab. 1), and the dorsal and anal fins begin to appear (Figs. 3G–I). No spines are observed on the body.

Flexion stage. Dorsolateral pigmentation of larvae intensifies in the flexion stage (5.89–7.98 mm BL), except for the posterior third of the body, which remains pigment-free (Figs. 3J–R). Dark pigmented cells are dispersed from the head to anterior insertions of the dorsal and anal fins, as well as along the lateral area of the body, with a concentration in the abdominal lateral region. In the ventral region, pigmentation is almost absent.

The dorsal and anal fin rays, which begin to develop at the end of the preflexion stage (marked by the initiation of notochord flexion), are fully formed by the completion of notochord flexion. At this stage, individuals with a body length of 5.69 mm or more typically have 8 to 9 rays in the dorsal fin and 9 to 10 rays in the anal fin (Tab. 1; Figs. 3M–P). The caudal fin rays are the last to develop, with a range of 8 to 11 rays (Tab. 1; Fig. 3P). Larvae lack body spines. The first observation of body inflation was recorded in a larva measuring 5.98 mm BL.

TABLE 1 | Morphological variables of Shoeroides pachygaster at larval stages. N = number of individuals; X = mean; SD = standard deviation; CF = caudal fin; DF = dorsal fin; AF = anal fin; and PF = pectoral fin.

S. pachygaster		Larval stage
		Preflexion (N = 13)		Flexion (N = 6)
		x̅ ± SD	Range	x̅ ± SD	Range
Measurements (mm)	BL	2.98 ± 0.77	1.54–4.16	6.10 ± 0.20	5.69–6.43
	BD	1.05 ± 0.24	0.60–1.42	1.95 ± 0.22	1.56–2.27
	BW	0.70 ± 0.19	0.37–1.00	1.94 ± 0.28	1.39–2.53
	PDL	1.71 ± 0.44	0.89–2.52	4.46 ± 0.56	3.76–5.25
	PAL	1.74 ± 0.44	0.81–2.28	4.63 ± 0.59	3.68–5.44
	HL	0.93 ± 0.28	0.43–1.35	2.57 ± 0.32	2.08–3.03
	SNL	0.23 ± 0.11	0.07–0.41	0.62 ± 0.10	0.46–0.80
	ED	0.44 ± 0.13	0.22–0.62	0.94 ± 0.14	0.76–1.10
	ID	0.55 ± 0.13	0.34–0.75	1.33 ± 0.16	1.10–1.52
Body proportions	BD/BL	0.36 ± 0.02	0.32–0.44	0.31 ± 0.03	0.27–0.35
	HL/BL	0.31 ± 0.02	0.24–0.35	0.42 ± 0.04	0.37–0.49
	ED/HL	0.48 ± 0.04	0.38–0.61	0.36 ± 0.02	0.34–0.41
	ID/HL	0.62 ± 0.12	0.48–0.86	0.52 ± 0.02	0.47–0.56
	PDL/BL	0.58 ± 0.04	0.46–0.65	0.73 ± 0.08	0.64–0.83
	PAL/BL	0.58 ± 0.03	0.52–0.66	0.76 ± 0.08	0.65–0.84
	BW/BL	0.24 ± 0.03	0.17–0.31	0.32 ± 0.04	0.24–0.39
Number of rays	PF	–	16–17	–	16–17
	DF	–	–	–	9–10
	AF	–	–	–	8–9
	CF	–	–	–	8–11

Growth patterns. In the preflexion stage, S. pachygaster larvae measured 1.53 to 4.16 mm in BL, with a moderately deep body (BD = 35.8% of BL), a moderately sized head (HL = 30.7% of BL), and large eyes (ED = 48.6% of HL) (Tab. 1; Figs. 4A–C). During the flexion stage, BL ranged from 5.69 to 6.43 mm. Body depth decreased slightly to 33.9% of BL, remaining moderately deep bodied, while the head became proportionally larger (HL = 42.1% of BL) and the eyes stayed relatively large (ED = 36.8% of HL) (Tab. 1; Figs. 4A–C).

FIGURE 4| Morphological proportions in relation to body length (BL, mm) during the early development of Sphoeroides pachygaster. Symbols: blue, preflexion stage larvae; red, flexion stage larvae. The gray area represents the 95% confidence interval for the fitted line. A. Body depth to body length ratio (BD/BL); B. Head length to body length ratio (HL/BL); C. Eye diameter to head length ratio (ED/HL); D. Interocular distance to head length ratio (ID/HL), E. Pre-dorsal length to body length ratio (PDL/BL); F. Pre-anal length to body length ratio (PAL/BL); and G. Body width to body length ratio (BW/BL).

The linear regression models indicate that BD/BL, ED/HL, and ID/HL ratios decrease from the preflexion to the flexion stage (Figs. 4A, C, D), while HL/BL, PDL/BL, PAL/BL, and BW/BL ratios increase from the preflexion to the late flexion stage (Figs. 4B, E–G).

Spatial distribution of Sphoeroides pachygaster (larvae and adult). We collected 4,094 larvae of S. pachygaster, with 4,088 in the preflexion stage and only six in the flexion stage. No larvae were found in the postflexion stage. We collected larvae during the austral winter and spring of 2019 (August and October), late spring of 2021 (December), and summer of 2022 (March). Sphoeroides pachygaster larvae were widely distributed throughout southeastern Brazil, occurring from the outer continental shelf (150 m isobath) to the offshore oceanic area, reaching to the 2,200 m isobath in the Santos Basin (red circles in Fig. 5).

FIGURE 5| A. Georeferenced distribution map of Sphoeroides pachygaster along the western South Atlantic; and B. Enlarged area (delineated in black) showing records of larvae (red circles) and adults (black circles) of S. pachygaster on the outer continental shelf of Southeastern Brazil. Adult sources: Figueiredo et al. (2002) and Bernardes et al. (2005). The abbreviations indicate Brazilian states: MG = Minas Gerais, ES = Espírito Santo, RJ = Rio de Janeiro, SP = São Paulo, PR = Paraná, SC = Santa Catarina, and RS = Rio Grande do Sul.

Adults of S. pachygaster have been recorded along the southeastern and southern coasts of Brazil, at depths of 50 to 200 m (black circles in Fig. 5). Additionally, three specimens were recorded at approximately 560 m depth off the northeastern Brazilian coast (Fig. 5A).

Vertical distribution. Larvae of S. pachygaster occurred throughout the surface to 550 m depth, with 99% collected in the first two layers (0–25 m: 75.5%; 25–50 m: 23.5%) (Fig. 6A). Preflexion larvae were almost exclusively found within the first 50 m (Fig. 6B), while flexion larvae were mostly found between 50 and 100 m, with occasional occurrence between 150 and 550 m (Fig. 6B).

FIGURE 6| A. Vertical distribution of the relative abundance of Sphoeroides pachygaster larvae in the Santos basin; B. Violin plot showing the proportion of S. pachygaster larvae at preflexion and flexion stages across different depth ranges (0–25, 25–50, 50–100, 100–150, 150–550, 550–1100, 1100–1500, 1500–2400 m). The width of the plot represents the proportion of each ontogenetic stage, and the numbers indicate the quantity of specimens in each depth stratum.

The estimated abundance (mean ± standard deviation) of larvae collected with 200 μm mesh nets was 9.95 ± 19.6 larvae.m^-3 in the upper layer (0–25 m), 2.99 ± 5.96 larvae.m^-3 in the 25–50 m layer, and 0.05 ± 0.05 larvae.m^-3 in the 50–100 m layer. Larval abundance in the 500 μm mesh nets was 0.03 ± 0.01 larvae.m^-3 in the 0–25 m layer, 0.02 ± 0.02 larvae.m^-3 in the 25–50 m layer, and 0.01 ± 0.01 larvae.m^-3 between 50 and 100 m layer.

Discussion​

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Here, we provide information on the larval development of S. pachygaster in the western South Atlantic, focusing on morphological and morphometric changes during early ontogeny. The molecular tools employed were effective in complementing species identification. Previous studies have applied integrated approaches combining morphology and DNA barcoding to identify fish larvae (Becker et al., 2015; Azmir et al., 2017; Silva-Segundo et al., 2021; Muguet-Julio et al., 2022; Pozzobon et al., 2024), as diagnostic traits based on morphology, meristic data, and pigmentation patterns can be difficult to interpret (Zhang, Hanner, 2012).

The phylogeny indicates that S. pachygaster is a sister group of the other species of Sphoeroides and suggests a basal position within the genus, a result similar to those obtained in previous studies (Amaral et al., 2013; Araújo et al., 2023; Hunt et al., 2023). Additionally, Sphoeroides and Lagocephalus showed a close phylogenetic relationship (~0.2). In contrast, the largest genetic distances (0.23) were observed between our sequences and those of Canthigaster figueiredoi and C. rostrata, which clustered into a distinct clade. Morphologically, Canthigaster larvae differ from Sphoeroides of similar size by their longer, more widely spaced dermal spines and the elongated, pigmentation and pointed snout typical of the genus (Richard, 2005).

The larval development of S. pachygaster has typical features found in other species of the Tetraodontidae, including tiny larvae (1.5 to 6.43 mm), a moderate to large-size head with a rounded profile, large eyes, a relatively small mouth with beak-shaped teeth, a small gill opening located below the middle of the pectoral fin, absence of pelvic fins, dorsal and anal fins positioned towards the posterior region of the body, and 18 vertebrae (Moser, 1996; Lyczkowski-Shultz, 2005).

Despite the diversity within the Tetraodontidae, knowledge of their larval development remains limited. Detailed descriptions, including meristic traits, morphology, and pigmentation patterns, are available for only a small number of species (Stroud et al; 1989; Byeon et al., 2011; Baldwin, 2013; Martínez-Brown et al., 2019; Syafiq et al., 2020). This scarcity of information hinders the accurate identification of larvae (Fricke et al.,2025).

The preflexion stage of S. pachygaster can be distinguished from that of other members of the Tetraodontidae by unique features and their distinct distributions. While Sphoeroides, Lagocephalus, Canthigaster, and species overlap geographically along the Brazilian coast, species such as Sphoeroides greeleyi Gilbert, 1900, S. testudineus (Linnaeus, 1758), S. spengleri (Bloch, 1785), S. dorsalis Longley, 1934, S. tyleri Shipp, 1972, Lagocephalus laevigatus (Linnaeus, 1766) and Canthigaster figueiredoi are primarily found in shallow waters, typically at depths below 100 meters (Shipp, 2002; Froese, Pauly, 2024). In contrast, S. pachygaster inhabits the outer shelf at depths exceeding 150 m (Figueiredo, Menezes, 2000; Figueiredo et al., 2002; Bernardes et al., 2005; Denadai et al., 2012; Froese, Pauly, 2024).

The larval pigmentation of S. pachygaster differs from Canthigaster spp., with melanophores concentrated dorsally and laterally but absent on the posterior third of the body. In contrast, Canthigaster larvae exhibit a pigmented stripe extending from the dorsal to ventral margins of the tail (Fig. 7C) (Arai, Fujita, 1988; Stroud et al., 1989; Sikkel, 1990). Additionally, S. pachygaster lacks dermal spines throughout its development (Figs. 7E–F), whereas larvae of Canthigaster, Lagocephalus, and other Sphoeroides spp. develop dermal spines in at least some body regions during larval development (Figs. 7A–D) (Fujita, 1966; Moser, 1996; Lyczkowski-Shultz, 2003).

FIGURE 7| Preflexion stage larvae of unidentified Tetraodontidae (A), with a close-up of the outlined area highlighting the presence of dermal spines (B, red arrows). Posflexion stage larvae of Canthigaster figueiredoi (C) and close-up showing dermal spines (D, red arrows). Flexion stage larvae of Sphoeroides pachygaster (E) and close-up of the outlined area, showing the absence of dermal spines (F).

The fin ray development in S. pachygaster follows the sequence: pectoral, dorsal, anal, and, finally, caudal fins. By the end of the flexion stage, dorsal, anal, and caudal fin rays are fully formed, with larvae displaying meristic traits distinguishing them from other species of Tetraodontidae (Tab. 2). Sphoeroides pachygaster is identified by 9 to 10 dorsal and 8 to 9 anal fin rays, while Lagocephalus spp., despite a similar pigmentation pattern, have 12 to 15 dorsal and 11 to 14 anal fin rays. Other Sphoeroides species typically have 7 to 8 dorsal and 6 to 8 the anal fin rays (Tab. 2). Cantigaster figueiredoi overlaps with S. pachygaster in fin rays count but differ inhabitat preference, inhabiting coastal zones at depths of 1 to 35 m (Moura, Castro, 2002).

TABLE 2 | Selected meristic characters in species belonging to the family Tetraodontidae whose adults or larvae occur in the southwestern Atlantic. References: a- Richards, 2005; b- Fahay, 2007; c- Froese, Pauly, 2024; d- Present study.

Species	Dorsal	Anal	Caudal	Pectoral	Vertebrate	Depth range (m)	References
Canthigaster figueiredoi Moura & Castro, 2002	9–10	9	11	15(16)	17	1–35	a, b, c
Lagocephalus laevigatus (Linnaeus, 1766)	14(12–15)	12(11–14)	11	17(15–19)	19(18)	10–180	a, b, c
Lagocephalus lagocephalus (Linnaeus, 1758)	14(13–15)	12(11–13)	11	14(13–16)	18	10–476	a, b, c
Sphoeroides dorsalis Longley, 1934	8	7	11	16(15–17)	17(16)	18–100	a, b, c
Sphoeroides greeleyi Gilbert, 1900	8(7)	7(6)	11	14–15(13–16)	17(18)	–	a, b, c
Sphoeroides pachygaster (Müller & Troschel, 1848)	9 (8–10)	8(7–9)	11	15–17	18	50–2200	a, b, c, d
Sphoeroides spengleri (Bloch, 1785)	8(7)	7(6–8)	11	13(14)	17(18)	1–70	a, b, c
Sphoeroides testudineus (Linnaeus, 1758)	8(7–9)	7(6–8)	11	15(13–16)	17–18(16–19)	1–48	a, b, c
Sphoeroides tyleri Shipp, 1972	8(7)	7(6)	11	15(14–16)	–	10–80	a, b, c

Sphoeroides pachygaster likely develop the ability to inflate during early flexion stages, as observed in a 5.98 mm SL larva. Among Tetraodontidae, this trait emerges at different stages, from preflexion to juvenile (Welsh, Breder, 1922; Leis, 1984; Lyczkowski-Shultz, 2003). The capacity to inflate is a critical survival adaptation, deterring predation through rapid body expansion (Mittal, Banerjee, 1976; Hertwig et al., 1992). The earlier onset in S. pachygaster may provide a survival advantage by offering defense during vulnerable early-life stages.

The wide distribution of S. pachygaster preflexion larvae suggests extensive spawning grounds in the western South Atlantic. Additionally, their presence throughout austral winter, spring, and summer suggests a prolonged reproductive period (Ragonese et al., 2001). The spatial overlap between adults and early larvae reinforces the link between larval occurrence and spawning sites. In general, larval distribution mirrors adult patterns, occurring in suitable habitats that support reproduction and provide essential food resources (Lasker, 1979; Cushing, 1990; Carassou et al., 2012; Leis et al., 2013).

Even though most adults of S. pachygaster have been recorded predominantly in the south-southeast Brazilian coasts, with only three records in the northeastern coast (Sampaio et al., 2001), it likely inhabits the entire Brazilian offshore Exclusive Economic Zone. It has the broadest geographic distribution among Sphoeroides (Shipp, 2002), spanning in tropical and temperate regions of the Atlantic, Indian, and Pacific oceans, as well as the Mediterranean (Cervigón et al., 1992; Cervigón, 1995; Carpenter, De Angelis, 2016; Figueiredo, Menezes, 2000).

In contrast to the deep habitat of adults (Matsuura, Tyler, 1997; Shipp, 2002), preflexion larvae predominantly inhabit the upper 25 m, while flexion larvae are observed deeper, between 50 and 100 m. This suggests an ontogenetic shift in vertical distribution, with larvae migrating to greater depths as they develop. However, the limited number of flexion larvae available prevents determining whether this shift is individual or species-specific behavior.

Marine fish larvae, especially during the preflexion stage, often aggregate in surface water due to the positive buoyancy of their eggs (Boehlert et al., 1992; Heath, 1992; Conway et al., 1997; Huebert et al., 2011). As sensory and swimming abilities develop, ontogenetic vertical shifts occur, enabling depth regulation (Huebert, 2008; Leis, 2010). Their vertical distribution and transition to deeper layers are influenced by factors such as food availability (Coombs et al., 1994; Job, Bellwood, 2000; Sassa, Kawaguchi, 2005), predator pressure (Lampert, 1989; Job, Bellwood, 2000; Huebert et al., 2011), and oceanographic processes (Nissling et al., 1994; Vilchis et al., 2009; Mora, Sale, 2002; Namiki et al., 2017).

As one of the deepest-dwelling pufferfish, S. pachygaster early larval distribution offers key insights into its ecological and evolutionary features. Additional larval data will enhance our understanding of its broad geographic distribution, spawning patterns, and developmental processes, supporting further research aimed at understanding the determinants of the larval distribution of S. pachygaster.

Acknowledgments​

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The authors are grateful to PETROBRAS for the coordination, execution, and funding for the “Santos Project – The Santos Basin Regional Environmental Characterization (PCR-BS)” under the RD&I investment clauses of the Brazilian National Agency of Petroleum, Natural Gas, and Biofuels (ANP). The Fundação de Apoio à Universidade de São Paulo (FUSP) managed financial resources that covered equipment purchase and maintenance, scholarship, travel, and other expenses. The Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) provided funds for the purchase of a Leica M205C stereomicroscope through the Multi-User Equipment Project EMU/FAPESP N^o 2017/04098–6. Special thanks to D. L. Moreira for cruise planning and project management and M. L. F. Rocha for assisting with sample registration and data management. We would like to express our sincere gratitude to S. Bonecker for her assistance with the molecular analyses. Petrobras provided funding through the RD&I investment clauses of the Brazilian National Agency of Petroleum, Natural Gas, and Biofuels (ANP). FUSP Project 3366.

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Authors

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Henrique Grande¹ , Ana C. T. Bonecker², Mariana M. Julio², Mario Katsuragawa¹, Maria de Lourdes Zani-Teixeira¹, Mateus G. Chuqui¹, Artur R. Spirgatis¹, Gabriela B. França¹, Frederico P. Brandini¹ and Cláudia A. P. Namiki¹

^[1]    Instituto Oceanográfico, Universidade de São Paulo, Praça do Oceanográfico, 191, 05508-120 São Paulo, SP, Brazil. (HG) henriquegrande@ymail.com (corresponding author), (MK) mkatsura@usp.br, (MLZT) zanit@usp.br, (MGC) mateuschuqui@gmail.com, (ARS) arturspirgatis@usp.br, (GBF) gabrielissima@usp.br, (FPB) brandini@usp.br, (CAPN) namiki@usp.br.

^[2]    Instituto de Biologia, Departamento de Zoologia, Universidade Federal do Rio de Janeiro – UFRJ, Bloco A, Ilha do Fundão, 21941 590 Rio de Janeiro, RJ, Brazil. (ACTB) ana@biologia.ufrj.br, (MMJ) marianamuguet@gmail.com.

Authors’ Contribution

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Henrique Grande: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing-original draft, Writing-review and editing.

Ana C. T. Bonecker: Data curation, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Mariana M. Julio: Data curation, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Mario Katsuragawa: Validation, Visualization, Writing-original draft, Writing-review and editing.

Maria de Lourdes Zani-Teixeira: Data curation, Writing-original draft, Writing-review and editing.

Mateus G. Chuqui: Writing-review and editing.

Artur R. Spirgatis: Data curation, Methodology, Writing-original draft.

Gabriela B. França: Data curation, Methodology, Writing-original draft.

Frederico P. Brandini: Funding acquisition, Project administration, Resources, Visualization, Writing-review and editing.

Cláudia A. P. Namiki: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing-original draft, Writing-review and editing.

Ethical Statement​

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The specimens were collected under a collection permit authorized by Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA processes ABIO Nº 1119/2019).

Competing Interests

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The author declares no competing interests.

How to cite this article

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Grande H, Bonecker ACT, Julio MM, Katsuragawa M, Zani-Teixeira ML, Chuqui MG, Spirgatis AR, França GB, Brandini FP, Namiki CAP. Molecular identification, early development and distribution of larval blunthead puffer Sphoeroides pachygaster (Teleostei: Tetraodontidae) off southeastern Brazil. Neotrop Ichthyol. 2025; 23(2):e240093. https://doi.org/10.1590/1982-0224-2024-0093

Copyright​

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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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© 2025 The Authors.

Diversity and Distributions Published by SBI

Accepted May 27, 2025

Submitted September 9, 2024

Epub August 04, 2025