Ovarian development and reproductive biology of Hoplias argentinensis (Characiformes: Erythrinidae), a top predator of the Pampa plain lakes, Argentina

Cristian Battagliotti¹ , Juan José Rosso^1,2 and Mariano González-Castro^1,2

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Abstract​

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Introduction​

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Although the Neotropical ichthyofauna has been studied for centuries, species diversity continues to increase rapidly, suggesting that current knowledge still underestimates actual diversity (Nelson et al., 2016). More than 6,400 species are recognized, but it is expected that there are nearly 9,000 (Facey et al., 2022). The family Erythrinidae (Characiformes) comprises 17 valid species and three genera, Erythrinus Scopoli, 1777, Hoplerythrinus Gill, 1896 and Hoplias Gill, 1903 (Toledo-Pizza et al., 2024). Based on morphology, three species groups can be recognized within Hoplias: H. lacerdae Miranda Ribeiro, 1908, H. malabaricus (Bloch, 1794) and the monotypic H. aimara (Valenciennes, 1847) (Oyakawa, Mattox, 2009). The H. malabaricus species complex has multiplied in recent years and consists of seven species: H. mbigua Azpelicueta, Benítez, Aichino & Mendez, 2015; H. misionera Rosso, Mabragaña, González-Castro, Delpiani, Avigliano, Schenone & Díaz de Astarloa, 2016; H. argentinensis Rosso, González-Castro, Bogan, Cardoso, Mabragaña, Delpiani & Díaz de Astarloa, 2018; H. auri Guimarães, Rosso, González-Castro, Souza, Díaz de Astarloa & Rodrigues, 2021; H. malabaricus, H. microlepis (Günther, 1864), and H. teres (Valenciennes, 1847).

In fish, the dynamics of the reproductive process and the viability of the offspring directly, though not exclusively, reflect the function of their gonads (Chaves, Vazzoler, 1984). Considering that the geographic distribution of each species is determined by a set of ecological conditions, it should have a single reproductive strategy and, to this end, specific anatomical, physiological, behavioral and energetic adaptations (Vazzoler, 1996). Nevertheless, reproductive variables reflect the adaptation of a given organism to the constraints imposed by the environment (Kjesbu, 2009; Brown-Peterson et al., 2011). Therefore, information on the reproductive processes (i.e., reproductive season, spawning season) of an individual species in its different geographical locations is important and necessary to support species management and conservation efforts. Hoplias argentinensis is the southernmost representative of the H. malabaricus group, widely distributed along the basins of Paraná-del Plata, Salado-Juramento, Salí-Dulce, Salado del Sur and Uruguay (Rosso, Liotta, 2021). It’s extremely austral occurrence within the genus and its wide geographical distribution make this species a good candidate for the study of important aspects of reproductive biology.

Although it is desirable to use data from both sexes when studying reproductive biology, there is a tendency to assume that females are more biologically informative than males (González-Castro et al., 2011; López et al., 2015). This is related to the fact that histological analysis of ovaries, when linked to the location and date of captured specimens, can accurately correlate spawning events with spatio-temporal (but also environmental) variables (González-Castro, Minos, 2016; Lajud et al., 2016). In addition, some important reproductive parameters such as fecundity are obviously only estimated in females. Finally, the duration of the spawning season of a population must be defined by the time that elapses between the beginning and the end of spawning events. It can only be measured by microscopic observation of the maturation stage of the ovaries, as it is known that males mature earlier and end their reproductive season after females (González-Castro, Minos, 2016; Heins, Brown-Peterson, 2022).

Reproductive effort, usually estimated with the Gonadosomatic Index (GSI) in fishes, indicates the proportion of total energy expended on physiological and behavioral aspects of reproduction, measured over a biologically significant period of time (West, 1990; Hutchings, 1993). Other body condition indices such as Fulton’s condition factor (K) (Ricker, 1975) are also routinely used to examine trends in weight gain in relation to fish size. As these body condition factors are largely influenced during the reproductive season (Marshall et al., 2004; Wuenschel et al., 2019), they provide a complementary approach for assessing population and individual performance during the reproductive season. Macroscopic descriptions of condition, size, color or other observable characteristics during ovarian development have been studied in the past for fisheries purposes (Clark, 1931). In turn, histological analysis of oocyte developmental stages has been an important tool for characterizing these macroscopic stages (West, 1990). The combination of histological analysis with macroscopic observations therefore provides adequate guidelines to characterize and detect additional information, such as the type of spawning pattern or fecundity (Heins, Brown-Peterson, 2022).

Hoplias argentinensis lives in small streams, canals, rivers and shallow lakes, generally in association with shallow areas and large mats of vegetation (Rosso, 2006; Rosso et al., 2018). The recent distinction of this species from H. malabaricus poses a major challenge for management and conservation measures, as both aspects need to be based on accurate knowledge of biological aspects of the species. There are some previous contributions on reproductive aspects, biological indices and growth patterns (Balboni et al., 2009, 2011) for populations of H. malabaricus from areas currently known to be inhabited by H. argentinensis. On the other hand, some macroscopic information on the reproduction of H. argentinensis was collected in an unpublished PhD thesis (Balboni, 2021). With some caution, this information could serve as a first guide to understand the basic biological characteristics of H. argentinensis. However, understanding the reproductive biology requires a study that includes a macroscopic assessment of the ovarian phases and subsequent histological examination of the same ovaries with precise terminology (Brown-Peterson et al., 2011; Heins, Brown‐Peterson, 2022). Although there are some reproductive studies on H. malabaricus (Marques et al., 2000; Gomes et al., 2007, 2015; Fernandes et al., 2021), all of them focus on the homonymous species. Unfortunately, the only available reference for microscopic reproductive aspects of the genus Hoplias is from a population labelled as H. malabaricus from southern Brazil (Matkovic, Pisano, 1989; Marques et al., 2000; Gomes et al., 2015).

In this context, the aim was to evaluate the life history pattern of H. argentinensis from a shallow, lacustrine environment of the southern pampas plain, Argentina. We tested the hypothesis that the reproductive season of H. argentinensis, occurs in late spring and summer, due to the higher temperature registered in these seasons. Our objectives were: (1) analyze the reproductive cycle as a function of environmental variables, (2) to perform a morphological/histological analysis of ovarian development and describe the developmental stages of the oocytes, and (3) to estimate the main bio-reproductive parameters (frequency of oocyte diameter distribution, batch fecundity, gonadosomatic index, Fulton’s condition factor) for the studied population of H. argentinensis.

Material and methods

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Study area. Lake Kakel Huincul (KH), 36°48’S 57°47’W, Argentine (Fig. 1) is an elongated, shallow body of water (2,000 ha, 0–4 m deep). At one end it is connected to Channel 2, which drains the water into Samborombón Bay. With no direct inflow from rivers or streams, its state is influenced by the annual rainfall variables themselves, most of which enters the system during floods by overflowing Channel 2. The shallow Lake KH is located in an area considered extremely humid: the southeastern Pampa plain (Aliaga et al., 2017). The climate is temperate with average annual rainfall of 987 mm and an average temperature of 15.1 °C, with the extremes for both variables occurring in January (109.2 mm and 28.5 °C) and July (62.1 mm and 3.8 °C) (National Meteorological Service, 2021–22). Originally, the shallow KH Lake was identified as a clear water environment (Allende et al., 2009) with abundant macrophytes. Nevertheless, in the last 25 years, the lake has undergone a process of eutrophication related to agricultural intensification (Sánchez Vuichard et al., 2023), changing its ecological status to a very turbid environment (Izaguirre et al., 2022).

FIGURE 1| Map of South America showing the exact location of Kakel Huincul lake and the nearby wetlands, Pampa Plain, Buenos Aires, Argentina (-36.8 S. -57.78 W). Photo (by Cristian Battagliotti) of vegetated littoral in Kakel Huincul, were most of the specimens were collected.

Fish and environmental sampling. Water temperature, water conductivity and dissolved oxygen were measured in situ with a Horiba U-53 multiparameter probe at a depth of 0.5 m during each sampling. Specimens of H. argentinensis were collected monthly between May 2022 and May 2023. Reference material for this species was deposited at the Fish Collection of the Instituto de Investigaciones Marinas y Costeras (UNMDP 5374; 5385–5387). Different fishing gears with complementary selectivity were used, including active (20 m long beach seine net, 10 mm mesh trawl) and passive (20 m long gill net and 110 to 120 mm mesh) techniques. Total length (TL) and standard length (SL) were determined to the nearest millimeter (± 0.1 cm). Total weight (TW) and ovary weight (OW) were determined to the nearest 0.1 g using electronic balance. The specimens were identified taxonomically according to Rosso et al. (2018).

Reproductive biology analysis. Ovaries were extracted in the field with a ventral incision, weighted in fresh, stored during 8 h in Davidson fixative solution, and preserved in ethanol 70% for the subsequent laboratory analysis. A piece of tissue from fixed and alcohol preserved ovaries was removed, dehydrated in ethanol 100%, cleared in xylol and embedded in paraffin. Sections were cut at 5 μm and stained with Harry’s hematoxylin followed by eosin counterstain as in González-Castro et al. (2011). The use of an appropriate terminology to describe teleost oogenesis allows and facilitates future comparisons (Heins, Brown-Peterson, 2022). Description of stages of oocyte development was performed following: Brown-Peterson et al. (2011), González-Castro, Minos (2016) and McMillan (2007). The histological classification of ovaries was based on oocyte development stage, and a macroscopic and microscopic ovary maturity scale of five phases was employed, both following Brown-Peterson et al. (2011). The frequency of distribution in the ovarian maturity phases was plotted bimonthly for greater representativeness of the data. Oocyte diameters were measured in each individual, a total of 220 oocytes per ovary were removed, placed in water, and the longest axis was measured with an ocular micrometer in order to check for the relation between oocyte size and oocyte developmental stage. The oocyte diameter frequency from subphase Active Spawning was plotted.

To gain a comprehensive understanding of the body condition of fish throughout the year, the Fulton’s condition factor (K) was calculated as K = TW × 100 / TL³, where TW: total weight, TL: total length, and the gonadosomatic index (GSI) was calculated as GSI = OW x 100/ (TW-OW), where OW: ovary weight (Ricker, 1975). Both were estimated employing cm and grams as length and weight unities, respectively. To compare indexes in relation to the annual cycle and between maturity phases, we used one-way ANOVA when the required conditions were fulfilled (Levene and Shapiro-Wilk) and Kurskal Wallis with no parametric data. All statistical analyses were performed with p value < 0.05 significance level. Reproductive indexes and environmental values were plotted together; the Fulton condition factor was multiplied by a factor of ten to a better visual understanding in the graph.

To estimate batch fecundity (number of oocytes released per spawning), ten ovaries in spawning capable maturation phase (actively spawning sub-phase), were used. These ovaries were characterized with final maturation oocytes and showed no evidence of recent spawning (no post ovulatory follicles were observed). Three portions (of approximately 0.1 g) of the anterior, medial and posterior parts of the gonad were rehydrated, weighed with an analytical balance (0.0001 g) and all final maturation oocytes were counted. Batch fecundity was estimated as the product between the mean number of yolked oocytes per ovary gram (Yo/g) and the Ovary weight (BF = Yo/g x OW). Relative fecundity (number of hydrated oocytes per gram of ovary-free body weight) was calculated as the batch fecundity divided by female weight (ovary-free) (Hunter et al., 1985). The relationship of batch fecundity to total length and to total weight, and relative fecundity to total length and to total weight were plotted and described using the Pearson correlation method (Kartas, Quignard, 1984).

Results​

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Fish and environmental sampling. During the study, extreme values of water temperature (8.3 to 31.2 °C) and water conductivity (4,340 to 9,730 µS/cm) were recorded while variation in dissolved oxygen (6.03 to 11.44 mg/l) were more moderate (Fig. 2A). A massive fish kill concomitant with extreme drought conditions in the region, extreme high-water temperatures (31.2 °C) and cyanophytes bloom occurred during late spring and early summer. Despite having multiplied the fishing effort very few individuals were collected during this period and no individual at all in January. Overall, a total of 40 females were collected and processed to be studied. Body size oscillates between 222 and 546 mm TL (mean 475 mm) and total weight from 138 to 2,333 g (mean 1,551 g).

FIGURE 2| A. Left axis: monthly mean water temperature (black triangles) and dissolved oxygen values (black circles), right axis: water conductivity (black squares). B. Monthly mean gonadosomatic index (GSI; black dotted line) and Fulton’s condition factor multiplied by ten (K; black solid line). *Refers to significant differences (p-value < 0.05). Obtained from Kakel Huincul shallow lake in 2022–2023.

Stages of oocyte development. The following stages of oocyte development were observed: primary growth oocyte (I and II), cortical alveolus stage, yolked (all sub-stages) oocytes, final maturation oocytes; also post ovulatory follicles and atretic follicle (alpha and delta) were recorded (Tab. 1; Fig. 3).

TABLE 1 | Descriptions of stages of oocyte development, atretic and post-ovulatory follicles of Hoplias argentinensis, obtained from Kakel Huincul shallow lake for the period of 2022–2023.

Stage (size-range)	Sub-stage	Microscopic characteristics
Oogonias (<50 µm)		Not found.
(A) Primary growth oocytes (50–200 µm)	I (Chromatin nucleus phase)	Dense nuclear granules centrally located in the nucleus with peripheral nucleoli. These cells had sparse cytoplasm and were surrounded by a limited number of squamous follicular cells.
	II (Perinucleolus phase)	Progressively, as the oocyte expanded, the nucleus enlarged to from the germinal vesicle and the cytoplasm exhibited decreased basophilic staining accompanied by discernible peripheral nucleoli. A count of sixteen to thirty-three nucleoli was noted.
(B) Cortical alveolus (250–450 µm)		Within the acidophilic cytoplasm, small empty vesicles were visible by hematoxylin-eosin staining. Simultaneously, the zona radiata surrounding the cell began to develop and became visible.
(C–D) Yolked oocytes (500–1200 µm)	Primary	It was characterized by enlargement due to the appearance of small acidophilic yolk globules. These globules disperse the cortical alveoli toward the central position. At the same time, the zona radiata, granulosa and follicular cells became prominently visible.
	Secondary	It was represented by an increase in the number of yolk droplets in the cytoplasm, an increase in the size of these droplets.
	Tertiary	The oocyte’s cytoplasm, along with the chorion or radiata zone, exhibited a complete vitellogenesis process, which was characterized by the presence of numerous yolk globules.
(E–F–G) Final maturation (1200–1950 µm)	Germinal vesicle migration (GVM)	The first visible event associated with final oocyte maturation was the migration of the germinal vesicle to the animal pole.
	Germinal vesicle breakdown (GVBD)	The envelope of the germinal vesicle breaks down and the nuclear contents merge with the surrounding cytoplasm.
	Hyalinization	Simultaneously, vitellogenin undergoes proteolysis, resulting in the release of free amino acids and promoting a homogeneous (hyaline) cytoplasm.
	Hydrated	Not found.
(H) Post-ovulatory follicles (POFs)		Residual follicular cells assume irregular shapes within the ovarian tissue. The follicular lumen decreased in size, while degradation intensifies proportionally with the time elapsed since ovulation.
(I–J) Atretic follicles	Alpha	Initial oocyte reabsorption, including any present yolk, occurs within the hypertrophied granulosa cells of the follicle.
	Beta	Not found.
	Gamma & Delta	Regression of the theca and granulosa cells persistently reduced the follicle size, accompanied by the appearance of yellowish-brown pigment.

FIGURE 3| Micrograph of the oocyte development of Hoplias argentinensis during the annual reproductive cycle throughout 2022–2023. A. Primary and secondary growth oocyte (PGI & PGII); B. Cortical alveolus stage oocyte (CA); C. Primary and secondary yolk stage oocyte (Vtg I & II); D. Tertiary yolk stage oocyte; E. Germinal vesicle migration, n: nucleus; F. Germinal vesicle breakdown; G. Hyalinization; H. Post-ovulatory follicle; I. Alpha atretic follicle; J. Gamma atretic follicle. Hematoxylin-eosin staining. Scale bars = 100 µm.

Macro and microscopic ovary maturity phases. Four of the five phases of gonad maturity were found throughout the sampled period (12 months): immature, developing, spawning capable (with subphase active spawning also recorded) and regressing (Fig. 4). The regenerating stage was not recorded.

FIGURE 4| Bi-monthly changes in the frequency distribution of ovary maturation phases in Hoplias argentinensis. The number of individuals sampled is indicated above each bar. Note the decrease in the number of individuals collected following the massive fish mortality recorded between December and February.

1. Immature (N = 4): Phase represented by oogonias smaller than 50 micrometers (µm) and primary growth oocytes. Ovaries small, transparent and thin with little connective tissue between the follicles and small space between oocytes in lamellae. Indistinguishable macroscopic blood vessels in the ovary (Fig. 5A). Average GSI: 0.19 (range: 0–0.3).

FIGURE 5| Macro- and micrograph of the phase of ovarian development in Hoplias argentinensis: macroscopic (left) and histological (right) images. A. Inmature phase. B. Developing phase. C. Spawning capable phase. D. Subphase active spawning. E. Regressing phase. Abbreviations correspond to: Primary growth oocytes (PG); Cortical alveoli oocyte (CA). Primary, secondary and third vitellogenic oocyte (Vtg, Vtg II, Vtg III). Post ovulatory follicles (POFs). Hematoxylin-eosin staining. Scale bars = 500 µm.

2. Developing (N = 7): This phase was constituted by primary growth oocytes, but also cortical alveolus, primary and secondary yolked oocytes stages (secondary growth oocytes) (Figs. 3A–C). Phase of gonadal growth and development that indicates the beginning of the reproductive season, previous to the spawning season. Medium sized, yellowish to slightly transparent ovaries, with a marked ovarian artery. The arterioles perpendicular to the ovarian artery were not notorious (Fig. 5B). Average GSI: 1.58 (range: 1–3.4).

3. Spawning capable (N = 16): In this stage prevailed oocytes with full vitellogenesis (Fig. 3D). Post ovulatory follicles may be present (Fig. 3H). Characterized by large, yellowish to orange, ovaries with prominent ovarian artery. Oocytes visible to naked eyes (Fig. 5C). Average GSI: 2.58 (range: 1.3–4.8).Active spawning subphase (N = 10): It was characterized by the presence of final-maturation oocytes (i.e., germinal vesicle migration (GVM), germinal vesicle break down (GVBD) or hydration) (Figs. 3E–G, 5D). Average GSI: 8.04 (range: 3–15.2).

4. Regressing (N = 3): This phase was characterized by the presence of numerous atresia and post ovulatory follicles (Figs. 3H–J). Flaccid, wasted ovaries, with prominent blood vessels. Soft white color. Primary growth oocytes are present, but also remaining yolked and/or hydrated oocytes can be found (Fig. 5E). Average GSI: 1.8 (range: 1–2.6).

Immature individuals were observed in May, while developing individuals were observed from May to September. The spawning capable phase was found from May to October, and its subphase active spawning were observed from October to February. The regression phase was found in April, corresponding to the end of the reproductive season before winter (Fig. 4).

Reproductive indexes. There were no significant differences in the Fulton condition factor across the months (Kruskal-Wallis = 0.430) or among the different ovary stages (f = 0.739, Tukey = 0.513). However, a slight decrease in this index was observed during the coldest months (May, June and July) (Fig. 2B). The GSI was significantly higher in October (p-value<0.05) with an average of 9 and the largest values reaching 15.2. According to the histological description and observed GSI values (>3), the spawning season begins in October and continues until February, despite the notorious rise in water temperature from 23 to 31 °C and the decay of dissolved oxygen (Figs. 2A–B, 4).

Frequency distribution of oocyte diameters. A total of 2,200 oocytes were measured. The size distribution of formalin-preserved oocytes during the active spawning sub-phase exhibited a multimodal pattern, which was histologically correlated with four different oocyte stages (Fig. 6). Four distinct modes were observed: the first mode, at 122 ± 54 μm, corresponded to primary growing oocytes; a second group at diameters of 316 ± 62 μm was indicative of cortical alveoli; a third mode at 723 ± 192 μm confirmed the presence of yolked oocytes; and finally, the last mode at 1,540 ± 174 μm denoted mature oocytes.

FIGURE 6| Frequency distribution of oocyte diameters of the reproductive subphase active spawning in Hoplias argentinensis. From black bars to white bars: primary growth oocyte, cortical alveoli oocyte, yolked oocytes and final maturation oocytes.

Fecundity. Batch fecundity averaged 54,650 oocytes/female with an estimated range between 24,342 and 77,866, corresponding to females of 497 and 533 mm TL respectively. No significant correlation coefficients were found between the batch fecundity, against TL and the TW ovary-free values (Figs. 7A–B). Relative fecundity has an average of 35.7 oocytes/female grams (free oocyte) with an estimated range of 12 to 67 oocytes per gram of female, for 497 and 533 TL, respectively. Again, no significant correlation coefficients were found (Figs. 7C–D).

FIGURE 7| Batch and relative fecundity graphs analyzed for spawning capable individuals of Hoplias argentinensis collected during the spring-summer season 2022–2023. A. Batch fecundity as function of total length. B. Batch fecundity as function of total weight without ovary. C. Relative fecundity as function of total length. D. Relative fecundity as function of total weight without ovary.

Discussion​

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Reproductive research within the Hoplias malabaricus species complex is primarily focused on the homonymous species H. malabaricus from Brazil (Marques et al., 2000; Gomes et al., 2007, 2015; Fernandes et al., 2021). Due to the need to constitute universal nomenclature to create a solid guide for future macroscopic reproductive research (Brown-Peterson et al., 2011), and according to the recent description of the argentine thraira H. argentinensis (Rosso et al., 2018), morphological/histological analysis of ovarian development were conducted. In addition, stages of oocyte development were described as also the main biological-reproductive parameters for the stock of H. argentinensis, in a typical shallow lake of the Pampa Plain, Argentine.

The immature phase of ovarian maturity, as defined by the presence of oogonias and primary growth oocytes (Brown-Peterson et al., 2011), was documented between the months of May and June. Although no oogonia nests were clearly distinguishable, the pool of primary growth oocytes ranged from 50 to 200 µm diameter and was recorded throughout the sampled period. These were followed by the next stages of oocyte development corresponding to cortical alveoli oocytes from 250 to 450 µm diameter. The presence of both, primary growth and cortical alveolus oocytes characterize the macroscopic developing stage (Brown-Peterson et al., 2011), being recorded in females collected from May to October. At the beginning of vitellogenesis, a decrease in cytoplasmic basophilia was observed, due to the intake of the yolk granules which possess a slight orange color because its affinity for eosin staining (Matkovik, Pisano, 1989; Vazzoler, 1996). The diameter of yolked oocytes ranged from 500 to 1,200 µm while the final maturation oocyte ranged from 1,200 to 1,950 µm. The mean diameter of mature oocytes observed in this study for H. argentinensis (1,540 ± 174 µm) is within the known size range for H. malabaricus. Mean diameter of mature oocyte in H. malabaricus can vary significantly, ranging from 1,222 µm (TL: 260 to 300 mm, Gomes et al., 2007) to 2,021 µm (TL: 311 to 439 mm, Gomes et al., 2015), with intermediate values of 1,410 µm (Oliveira, Nogueira, 2000) and 1,750 µm (Prado et al., 2006). Interestingly, the observed values in the studied population are very similar to those reported (1,522 ± 230 µm) for the large blue thraira H. lacerdae (Gomes et al., 2007). Multiple factors can affect the final size of eggs. The Thorson-Rass’s rules are used in marine animals to explain the inverse relationship between fecundity and egg size with temperature. Populations at higher latitudes begin to produce larger eggs due to phenotypic plasticity in colder environments (Laptikhovsky, 2006). While it is difficult to prove this change in a freshwater environment, there is evidence of adaptability to environmental conditions across latitudes in walleye fish (Johnston, Leggett, 2002) as well as in other fish species (McGurk, 2000; Tamate, Maekawa, 2000). Therefore, it is interesting to hypothesize a correlation between latitude and oocyte size at final maturation and/or total length of specimens due to phenotypic plasticity in response to colder environments and wider seasonality. In fact, previous work that began to look at karyotypic variation within the cryptic H. malabaricus complex had already suggested that lake populations should be managed as independent management and conservation units (Jacobina et al., 2011). Moreover, recent studies have linked karyomorphs, which could be H. argentinensis, in areas not previously thought possible for this species (lower basin of the Iguazú River) (Perin et al., 2024). This again demonstrates the biological and social interest of research in the H. malabaricus species complex with comparative studies according to species and region.

The presence of full vitellogenic oocytes characterized the spawning capable phase of the thrairas. It was recorded from May to February (but increasing in proportion from spring onwards), indicating that this period comprises the reproductive season. According to Brown-Peterson et al. (2011), the spawning season starts only when oocytes are present in their final maturation stage with germinal vesicle migration. The active spawning subphase was observed from October to February, which coincided with the highest recorded values for temperature and precipitation, as well as an increase in the GSI. A similar onset of the spawning season for H. argentinensis was reported for the Yalca lake, another typical shallow lake of the Pampa Plain in the lower Salado River basin (Balboni, 2021). Our microscopic approach allowed us to differentiate between spawning and reproductive periods and to note that the reproductive period ended in April with the regression phase. In fact, a slight decrease in the Fulton condition factor was recorded after this time, from May to July, which coincided with the lowest level of GSI. The decrease in temperature and the peak sun hours per day during those months in Southern Hemisphere also may help to explain this pattern by triggering a decrease in estrogens and progestogens, which generates at the oocyte level atretic follicles and reabsorption of unreleased oocytes. At the same time, the theca and granulosa cells hypertrophy facilitating the resorption of follicular tissues (González-Castro, 2016; Mokhtar, 2025), as was observed in the regression stage of this study. Overall, this decreases in reproductive activity with its adjustment in metabolism causes fish accumulating reserves for the following season. In this sense, possible thermal increases in lakes of the Pampa Plain during the warming seasons (such as those evidenced by Elisio et al., 2015), could lead to a metabolic imbalance of the species. Therefore, further research on the life history of local fishes is mandatory to understand and anticipate population variations.

Atretic follicles were observed in spawning capable phase, active spawning subphase and most frequently in regressing phase. According to Corriero et al. (2021), the atretic follicles observed in this study corresponded to the initial (alpha) and final (gamma and delta) stages. Hoplias malabaricus is known to possess extensive plasticity in the face of environmental variables such as pollution and is usually evidenced through the study of atretic follicles (Gomes et al., 2015). The presence of atretic follicles is described for the first time in H. argentinensis, a member of the H. malabaricus group, and opens opportunities for further research on the recovery capabilities of this species amid cyclical environmental variables.

Batch fecundity (BF) and relative fecundity (RF) were estimated with final maturing oocytes identified and measured by microscopic observation (diameter greater than 1250 µm). In comparison with populations of the H. malabaricus from Brazil, both batch and relative fecundity observed in this study (BF: 54,650 ± 21,543 oocytes/female; RF: 35.7 ± 17.7 oocytes/female grams (free oocytes) with TL 465 to 533 mm) were higher than previous one (BF: 9,620, with TL: 301 to 359 mm, Querol et al., 2003; BF: 8,472 ± 2,832, RF: 13.42, with TL: 316 to 414 mm, Gomes et al., 2015). At the same time, it must be noted that the total length of H. argentinensis is longer than the homonymous H. malabaricus. Taxonomic differences in the development and size of oocyte are reported. Irrespective of that, the batch fecundity of H. argentinensis of the KH lake is still markedly higher than values reported for the same species from a similar environment (BF: 11,432 ± 4,414 and RF: 20.73 with TL ranging from 284 to 600 mm, Balboni, 2021). While some studies calculate the oocyte size and fecundity from indirect volumetric data, others estimate it directly from the egg, overestimating the mean oocyte stage of final maturation. This study highlights manual counting from direct observation with ovary sub samples as a measurement fecundity method. Species of the H. malabaricus complex are multiple spawners with parental care and nest building (Prado et al., 2006). In multiple spawning species, fecundity estimates the number of oocytes released in each lot, and the number of vitellogenic oocytes can vary throughout the year depending on prevailing environmental variables (Rizzo, Bazzoli, 2020). A higher fecundity in a shallow lake with a summer season characterized by harsh environmental conditions imposed by drought and a cyanophyte bloom, as seen in the KH shallow lake, could be related to an adaptive strategy to periods of drought. However, to avoid possible bias in reproductive studies, by counting secondary growth oocytes or using incorrect developmental stages, previous established ovarian phases as shown here is necessary to an accurate fecundity measurements (Heins, Brown-Peterson, 2023).

The thraira is known for its ureotelic nature and remarkable adaptability to a wide range of pH levels, as well as its resistance to variable periods of anoxia or hypoxia (Rios et al., 2006). A massive fish kill was recorded in the KH shallow lake throughout our field sampling during the summer of 2023. The highest temperature, lower dissolved oxygen and higher water conductivity values recorded during the months of December, January, February and March concomitant with a severe regional drought, were followed by cyanophyte bloom and lately fish mortality. Although this environmental scenario is common in Pampa Plain aquatic ecosystems (Soria et al., 2008; Naya et al., 2011; Cocciolo et al., 2021), it was already warned that toxic cyanophyte blooms are expected to increase due to climate change and eutrophication (Aguilera et al., 2018; O’Farrell et al., 2021).

The H. malabaricus complex has a wide distribution, with three recently described species (Azpelicueta et al., 2015; Rosso et al., 2016, 2018) supporting important Argentine fisheries. Nowadays Hoplias spp., evidence declines in fish populations surveys in both the middle Paraná and the upper Paraná River, and in commercial fishery monitoring in Santa Fe, Argentina (Scarabotti et al., 2021). Traditionally, only a single species of Hoplias was recognized in Argentina. At present, there are three different species with a lack of the reproductive ecology associated in it. Poor or incomplete scientific knowledge of the bio-ecological characteristics of fishes is one of the main constraints and limitations observed in the management and conservation of freshwater fishes (Barletta et al., 2010). At the same time, recognizing different phases of ovarian development allows for understanding physiological adaptations, if any, to the particular environment of each population (Mendez et al., 2024). Therefore, studying ovarian stages over an annual period and correlating them with environmental variables will provide a fundamental tool for the correct management of these resources and aim to elucidate the evolutionary ecology of the species in different geographic areas that may have been apparently isolated thousands of years ago.

Acknowledgments​

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The authors are deeply grateful with Lic. Graciela Alvarez from Instituto Investigaciones Marinas y Costeras (IIMyC) for performing the histological section utilized in this study. We extend our appreciation to Mr. Marcelo Aceval, commissioner of the Kakel Huincul shallow lake, for generously granting access to the lake as well as offering assistance during our research activities. Also, Dr. Nicolas Chiaradía (technician of IIMyC) for logistic support in field sampling. We also wish to express our gratitude for the financial support received through the National Agency for the Promotion of Research, Technological Development, and Innovation. This work was supported by ANPCyT (PICT 2019–01344), CONICET, and Universidad Nacional de Mar del Plata (EXA 1069/22).

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Authors

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Cristian Battagliotti¹ , Juan José Rosso^1,2 and Mariano González-Castro^1,2

^[1]    Grupo de Biotaxonomía Morfológica y Molecular de Peces (BIMOPE), Instituto de Investigaciones Marinas y Costeras (IIMyC CONICET-UNMdP), Universidad Nacional de Mar del Plata, Dean Funes 3350 (7600) Mar del Plata, Buenos Aires, Argentina. (CB) cristianbattagliotti@mdp.edu.ar (corresponding author), (JJR) plurosso@yahoo.com.ar, (MGC) gocastro@mdp.edu.ar.

^[2]    Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mar del Plata, Argentina

Authors’ Contribution

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Cristian Battagliotti: Data curation, Formal analysis, Investigation, Software, Writing-original draft, Writing-review and editing.

Juan José Rosso: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing-original draft, Writing-review and editing.

Mariano González-Castro: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing-original draft, Writing-review and editing.

Ethical Statement​

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Collected fishes were immediately immersed in a saturated benzocaine solution (1 g/l) in accordance with the recommendations of good practice protocols in aquaculture and fisheries (Barker et al., 2002), and as approved and revised by the local committee for animal use and care in research (Comité Institucional para el Cuidado y Uso de Animales de Laboratorio, CICUAL, FCEyN-UNMdP, RD 126–18).

Competing Interests

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The author declares no competing interests.

How to cite this article

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Battagliotti C, Rosso JJ, González-Castro M. Ovarian development and reproductive biology of Hoplias argentinensis (Characiformes: Erythrinidae), a top predator of the Pampa plain lakes, Argentina. Neotrop Ichthyol. 2025; 23(2):e240109. https://doi.org/10.1590/1982-0224-2024-0109

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 April 3, 2025

Submitted October 27, 2024

Epub June 30, 2025