First evidence of gastric brooding in Asterinidae highlighted by the reproductive strategy of Asterina fimbriata in Atlantic Patagonia

First evidence of gastric brooding in Asterinidae highlighted by the reproductive strategy of Asterina fimbriata in Atlantic Patagonia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First evidence of gastric brooding in Asterinidae highlighted by the reproductive strategy of Asterina fimbriata in Atlantic Patagonia Ariana Belén Alarcón Saavedra, Sol Aylén Rebolledo, Gregorio Bigatti, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8451161/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The reproductive biology of Asterina fimbriata , a member of the family Asterinidae, was investigated through monthly sampling between July 2021 and June 2023 in Atlantic Patagonian coastal waters. This species presented a curious reproduction strategy: females exhibited an aperiodic reproductive pattern, characterized by the continuous presence of oocytes at different developmental stages co-occurring within the gonads and a low frequency of mature oocytes along the year. However, 6.52% of females exhibited a periodic oogenic cycle, showing a release maximum peak between May and September (coldest water temperature) Males displayed a clear annual gametogenic cycle, with maximum sperm release during the coldest months (June-August). Only 8% of females exhibiting brooding behaviour, which lasted approximately four months (May-September) and coincides after the major oocyte release season. Broods developed within the stomach and, following metamorphosis, emerged as a mass outside the female's mouth. Six distinct developmental stages were identified, revealing a progressive transition from internal to external brooding as offspring matured. A. fimbriata represents the first confirmed case of gastric brooding in the family Asterinidae. The low proportion of reproductive females would be indicating a high energetic cost of incubation that could be associated to the feeding behaviour of this fragile species. These findings expand the understanding of reproductive strategies in sea stars and highlight a novel adaptation within this clade. Sea stars South Atlantic Ocean Reproduction Development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Echinoderms exhibit a wide diversity of reproductive strategies. In general, sea stars from cold and temperate waters display cyclical reproductive cycles, often characterized by annual or seasonal patterns (Mercier and Hamel 2009 ; Cossi et al. 2015 ). These cyclical reproductive patterns are closely linked to environmental factors, such as temperature and photoperiod, which fluctuate according to predictable seasonal regimes (Mercier and Hamel 2009 ). However, some warm-water or deep-water species exhibit a continuous reproductive pattern associated with relatively stable environments, where environmental conditions remain largely unchanged (Byrne et al. 1997 ; Benítez-Villalobos et al. 2007 ; Mercier and Hamel 2009 ; Rivadeneira et al. 2017 ). Interestingly, seasonal reproductive patterns have also been reported in species traditionally considered to have continuous reproduction, including deep-waters species (Tyler et al. 1990 ) and shallow-water species from cold environments (Brogger et al. 2013 ). Mercier and Hamel ( 2009 ), along with other researchers, suggest that the term “aperiodic” is more appropriate than “continuous” when referring to the absence of a detectable reproductive pattern. In species with cyclic reproduction, particularly those with annual periodicity, gametogenesis typically follows a clear, synchronous, and orderly sequence of developmental stages, as documented in several species (Carvalho and Ventura 2002 ; Gil and Zaixso 2007 ; Gil et al. 2020 ). In contrast, species with an aperiodic reproductive pattern present greater challenges in identifying distinct developmental stages, as gametes at various stages of maturation are simultaneously present within the gonads (Byrne 1996 ; Brogger et al. 2013 ; Fraysse et al. 2020 ). Sea stars exhibit diverse modes and strategies of reproduction, ranging from broadcast spawning to species that incubate and brood their offspring (Byrne et al. 2003 ; Byrne 2006 ; Gillespie and McClintock 2007 ). The latter strategy is less common and has evolved independently within Asteroidea (Emlet 1990 ; Chia and Walker 1991 ; Lawrence and Herrera 2000 ). Brooding is typically found in certain families, such as Ctenodiscidae (Rivadeneira et al. 2017 ), Pterasteridae (Fraysse et al. 2020 ), Asteriidae (Pérez et al. 2017 ; Rivadeneira et al. 2020 ), and Asterinidae (Byrne 1996 ). This reproductive strategy appears to be characteristic of species inhabiting high and mid-latitudes (Mileikovsky 1971 ; Himmelman et al. 1982 ; McEdward 1995 ; Bosch and Slattery 1999 ; Fraysse et al. 2018 ). Furthermore, brooding strategies may occur within the parent’s body, such as inside the gonads, as observed in Patiriella vivipara , P. parvivipara , and Crysptasterina hystera (Byrne 1996 , 2005 ), or inside the stomach, as seen in Smilasterias multipara (O’Loughlin and O’Hara 1990 ). Other species possess specialized brood chambers located in different parts of the parent’s body, including the aboral and oral surfaces, as well as beneath the body. Examples include Ctenodiscus australis (Rivadeneira et al. 2017 ), Diplopteraster verrucosus (Fraysse et al. 2020 ), Anasterias minuta (Salvat 1985 ; Gil et al. 2011 ), and Anasterias antarctica (Pérez et al. 2017 ). During brooding, some females can continue feeding while incubating their offspring. For instance, individuals of Anasterias rupicola have been observed feeding while brooding (Blankley and Branch 1984 ). Similarly, Leptasterias littoralis can consume particulate material during this period (O’Brien 1976 ). Other species, such as Leptasterias pusilla and L. tenera , have been found with digested material in their mouths while brooding (Osterud 1918 ; Smith 1971 ; Hendler and Franz 1982 ). In contrast, some sea stars do not feed while brooding, including Anasterias minuta (Gil et al. 2011 ), A. antarctica (Pérez et al. 2017 ), and Leptasterias hexactis (Chia 1966 ). Understanding whether brooding females feed or not is essential to assess the energetic investment associated with this reproductive strategy. The family Asterinidae exhibits a variety of life histories (Dartnall 1971 ; Byrne and Cerra 1996 ; Byrne 2006 ), and within the order Valvatida, brooding has been reported in several species (Bosch and Pearse 1990 ; Byrne 2006 ; Pearse et al. 2009 ), particularly within the genera Asterina , one of the richest genera in terms of reproductive strategies (Rowe and Gates 1995 ). The genus Asterina comprises more than 30 species worldwide, of which only two, A. stellifera and A. fimbriata , have been reported in Argentina (Bernasconi 1973 ; Meretta et al. 2016 ). Asterina stellifera is larger than A. fimbriata and is distributed from Cabo Frio, Brazil (23°S, 42°W), to Mar del Plata, Argentina (38°S, 56°W), in the southwestern Atlantic Ocean (Clark and Downey 1992 ). This species follows a synchronous reproductive cycle and releases its gametes into the water column (Carvalho and Ventura 2002 ). In contrast, A. fimbriata is a smaller sea star with a moderately flattened, pentagonal body, displaying limited differentiation between its arms and central disc (Bernasconi 1973 ). This species has been recorded throughout southern Patagonia (Argentina and Chile), from approximately 41° S southward to the Magellanic region, including Camarones Bay (Chubut, Argentina), the Beagle Channel, and Macrocystis kelp forests located south of the Strait of Magellan. It is present in both the Atlantic and Pacific sectors and occurs from intertidal zones down to depths of nearly 300 meters, inhabiting various substrates such as rock, gravel, sand, mud, and kelp (Bernasconi 1973 ; Clark and Downey 1992 ). Currently, knowledge regarding the biology of this species remains scarce. The present study investigates the reproductive biology of Asterina fimbriata through monthly histological analyses of the gonads and by describing its brooding behaviour. This includes the characterization of offspring developmental stages, examination changes in brood size, and identification of morphological variations. Additionally, the study discusses the reproductive strategy of brooding in relation to established patterns observed in other sea star species, highlighting the associated advantages and disadvantages in terms of energy investment and feeding behaviour during the brooding period. 2. Material and methods 2.1 Study area and field sampling Between 20 and 30 specimens of Asterina fimbriata were collected monthly by SCUBA diving at depths of 4 to 8 m in Camarones Bay, Chubut, Argentina (44°46'S, 65°34'W), from July 2021 to June 2023. Most specimens had a radius greater than 5 mm, though a few smaller individuals were found on hard rocky substrates covered with various algae and kelp forests, primarily Undaria pinnatifida and Macrocystis pyrifera . Asterina fimbriata typically inhabits numerous cavities and crevices within the rocks, which provide shelter. This locality is a bay with open waters to the sea (Schillizzi et al. 2014 ) with an attenuation coefficient ( K d ) ranging from 0.25 to 0.31 m − 1 (Villafañe et al. 2004 ). In the field, all specimens were fixed in Bouin’s solution for 48 hours and subsequently transferred to 70% ethanol. Each specimen was measured using a Vernier calliper to determine the radius (R), defined as the distance from the centre of the disc to the tip of the longest arm, and the radius (r), defined as the distance from the centre of the disc to the interradius. The gonads were then dissected and preserved for histological analysis. 2.2 Sex determination and histological analysis For histological analysis, the gonads were dehydrated in graded ethanol solutions and embedded in paraffin. Sections of 5–6 µm thickness were cut and stained with haematoxylin and eosin. The diameter of each oocyte, with a distinct nucleolus, was measured as the mean value between the maximum diameter and its perpendicular at the oocyte’s centre (McClary and Mladenov 1989 ; Byrne 1992 ; Bosch and Slattery 1999 ). Measurements were performed using AxioVision SE64, Rel. 4.9.1 software, and gonad sections were photographed under a Stemi 2000-C stereomicroscope and a ZEISS Axiocam ERc5s camera. For sex determination, gonads were examined microscopically, and sex was confirmed through histological analysis. A Chi-squared test was applied to assess significant differences in sex ratios throughout the study period and for each month individually. The seawater temperature and depth were recorded using a dive computer. The pH was measured using a “Milwaukee” pH meter, and salinity was measured with a field ATC refractometer. All parameters were recorded monthly in situ in each sampling. The monthly daylight average (photoperiod) was acquired from the Servicio de Hidrografía Naval ( 2024 ). We related the oocyte diameter measurements from cells with a distinct nucleolus in all females with environmental parameters to estimate the gonadal cycle as was done by Nieto Vilela et al. ( 2019 ) and Cumplido et al. ( 2022 ). 2.3 Brooding and developmental stages All specimens were thoroughly examined to identify different stages of brood development inside the parent’s body and oral cavity, using a stereoscopic magnifier. Offspring were counted, measured, and photographed. Various terms have been used in the literature to denote incubating parents, embryos, young, juveniles, and offspring (see: (Byrne et al. 2003 ; Gil et al. 2011 ; Byrne 2013 ; Rivadeneira et al. 2017 ; Fraysse et al. 2020 ). In this study, the term “broods” refers to offspring produced and cared for by a single parent, while “brooding females” strictly refers to females exhibiting brooding behaviour. 3. Results 3.1 Sex determination Of the 572 individuals of Asterina fimbriata studied, 276 were females, 292 were males, and 4 were immature. The sex ratio of the population did not differ significantly from 1:1 (χ² = 0.47, p > 0.05), nor did it differ significantly for each month (p > 0.05). The R ratio in the A. fimbriata population ranged from 4.15 to 14.86 mm (mean = 8.97 mm, SD = 1.46 mm), while the r ratio varied from 2.98 to 10.12 mm (mean = 6.40 mm, SD = 1.04 mm). We found that the four smallest individuals (R < 5 mm) lacked gonads or developing gametes and were therefore excluded from the analysis. 3.2 Histological analyses Histological analysis was conducted for 260 females and 279 males. Each individual presented five pairs of gonads, located in the interradius on the aboral side of each arm. Ovaries were spherical and orange/yellow, while testes were branched with tubular structures and white in colour. The gonad morphology comprised two sacs: an outer sac of connective tissue and an inner sac of germinal cells, separated by a basal membrane. No notable changes in gonad external morphology were observed in females throughout the study period. 3.2.1 Females Oocytes were classified into the following categories: previtellogenic oocytes , small, with thick walls, a distinguishable nucleolus, and a large nucleus; intermediate oocytes : characterized by the presence of some yolk granules and thinner walls; and mature oocytes : filled with granules and lipid droplets, with thin walls. All females, including both brooders and non-brooders, exhibited ovaries containing oocytes at various developmental stages simultaneously (Fig. 1 ). Previtellogenic (< 100 µm) and intermediate (100–400 µm) oocytes were the most abundant across all months. We identified structures compatible with certain gonadal stages in females each month through histological analysis. Phagocytes were observed in 40% of females throughout the study period (Fig. 1 A). Developing ovaries containing oogonia, previtellogenic, and intermediate oocytes were also observed, although a pattern was not consistently observed across months. Only 18 females sampled had partially spawned ovaries during the periods from July to September 2021 and from November to December 2021, as well as from May to September 2022, from November to December 2022, and in June 2023. These ovaries were characterized by empty spaces in the lumen and the presence of some vitellogenic and previtellogenic oocytes (Fig. 1 B, D). Accumulation of mature oocytes (> 400 µm) was detected only in 17 females (Fig. 1 B). 3.2.1.1 Oocytes size frequency distribution We measured 6,011 oocytes with a distinct nucleolus, registering an average of 6 to 64 oocytes per female. The minimum oocyte diameter recorded was 17.82 µm (September 2021), while the maximum diameter was 916.31 µm (April 2023). Frequency histograms showed that the most common oocyte sizes ranged from 50 to 200 µm, indicating a constant presence of early-stage oocytes throughout all months (Fig. 2 ). Although no significant accumulation of large oocytes (550–600 µm) was detected, histograms showed a low frequency (< 10%) of these oocytes during the study period. The constant presence of early-stage oocytes throughout all months corresponded with the growing stage observed in females (Fig. 3 ). In addition, the occurrence of large oocytes coincided with the presence of mature stage in some females, while their subsequent disappearance accorded with periods in which spawning females were recorded (Fig. 3 ). In order to discover the influence of environmental variables in females’ gonadal cycle, we noted that water temperature and day length followed a clear annual cycle, increasing during austral summer (December-February) and decreasing during austral winter (June-August) (Fig. 4 ). The temperature reached a maximum of 17.5ºC in February at 4.5 meters depth, while day length reached a maximum of 15.53 h of light in December (Fig. 4 ). Both variables were positively correlated (Pearson = 0.76). Oocyte diameter showed weak fluctuations throughout the study period, with no strong seasonal trend. In addition, there was no correlation between oocyte diameter and day length (Pearson = 0.01) or temperature (Pearson = 0.19). However, in both 2022 and 2023, a mainly peak in mean oocyte diameter (> 250–300 µm) was observed during April –May, once environmental variables had already begun to decline. These peaks were followed by a decrease from June, indicating release of mature oocytes during the austral autumn–winter. 3.2.2 Males In contrast to females, defined gonadal development stages were identifiable in males through histological analysis. Testes were classified into the following stages: 1) Growing stage : characterized by a spermatogonia layer resting on the inner sac, spermatic columns composed of spermatocytes, and some spermatozoa in the lumen (Fig. 5 A). 2) Mature stage : testes had slim walls, a thin spermatogonia layer, shorter spermatic columns, and a lumen filled with spermatozoa (Fig. 5 B). 3) Spawning stage : testes exhibited thick walls, few spermatogonia, empty spaces, and residual spermatozoa in the lumen, occasionally accompanied by phagocytes (Fig. 5 C). 4) Post-spawned stages : characterized by the presence of spermatogonia predominantly in the lumen, occasionally accompanied by phagocytes (Fig. 5 D). 5) Resorption stage : marked by phagocytes predominantly in the lumen, occasionally alongside spermatogonia (Fig. 5 E). We noted that spermatogonia were present throughout the study period, even in mature males, becoming more evident during the post-spawning stages. Moreover, testes contained multiple developmental stages simultaneously, and mature and spawning testes were occasionally observed within the same individual (Fig. 5 F). 3.2.2.1 Temporal trend of testicular development The temporal analysis of testicular development indicated an annual gametogenic cycle (Fig. 6 ). The growing stage was predominantly observed during the austral summer (December and January), when temperatures ranged 15–16ºC. The mature stage occurred mainly from the austral summer to mid-autumn (February to May, including June 2023), when temperatures ranged 17–10ºC. The spawning stage was predominantly observed during the austral winter (from June to August), when temperatures ranged approximately 10–8ºC. The post-spawning stage was mainly observed during austral spring (from September to November), when temperatures ranged 9–11ºC. Finally, testes in resorption stage were observed throughout the year, with a higher incidence (10%–30%) from March to November 2022. 3.3 Brooding behaviour and developmental stages We studied three brooding seasons from July 2021 to June 2023, identifying 2 to 5 brooding females per month between May and September, totalling 23 brooding females, representing 8% of the total females studied. The R ratio in brooders ranged from 8 to 12.7 mm (mean = 9.48 mm, SD = 1.42 mm), and we recorded 505 broods, with 5 to 99 broods per female (Table 1 ). Most broods were located in a mass outside the female’s mouth, although some were found inside the females. Table 1 Developmental stages of broods recorded in Asterina fimbriata from July 2021 to June 2023. The table shows the number of broods per female and the corresponding developmental stages observed in each brooding female per sampling month. Date Number of broods Developmental stages observed 2021 07/2021 15 II-III-IV 07/2021 5 III-IV 08/2021 9 IV-V 08/2021 41 IV-V 09/2021 10 VI 09/2021 6 V-VI 2022 05/2022 1 I 05/2022 54 I 05/2022 99 I 06/2022 47 I-II 07/2022 15 IV-V 09/2022 20 VI 09/2022 29 V-VI 09/2022 17 IV-V 09/2022 34 II-IV-V 09/2022 27 V-VI 2023 06/2023 5 II 06/2023 15 I-II 06/2023 28 II-III-IV 06/2023 16 I 06/2023 12 I-II Early developmental stages were located in the stomach (Fig. 7 A, B), intermediate stages were found near the mouth (Fig. 7 C), and fully developed broods were observed in a mass outside the mouth, beneath the females' arched arms (Fig. 7 D). Although a general spatial pattern of brood development was observed, with broods progressing from the stomach toward the mouth as development advanced, this pattern was not always strictly followed. In some females, broods at different developmental stages were found co-occurring within the same anatomical region, including more advanced stages still in the stomach or earlier stages near the mouth (Table 1 ). We identified six developmental stages, classified by size and morphological characteristics (Fig. 8 ). Stage I : (Mean diameter: 929 µm; N: 121). Broods located in the stomachs of females. They were spherical or oval, bright yellow, surrounded by a transparent membrane, with no visible structures (Fig. 8 A). Stage II : (Mean diameter: 1154 µm; N: 104). Broods still located in the females' stomachs, but they adopted an oval and elongated shape, surrounded by a transparent membrane. Some broods exhibited a depression in the middle of the body, while others displayed two depressions at opposite extremes (Fig. 8 B). Stage III : (Mean diameter: 1146 µm; N: 48). Broods still located in the females' stomachs. Incipient tube feet emerged through the body wall, indicating the future formation of the ambulacral groove and the oral surface (Fig. 8 C). Stage IV : (Mean diameter: 1174 µm; N: 68). Broods peeping from the females' mouths, visible externally. Body of broods with pentagonal shape, with two pairs of tube feet per radius (Fig. 8 D). Stage V : (R: 756 µm; N: 90). Broods located in a mass outside the females' body on the oral surface. Adult-like shape, with ambulacral grooves distinguishable, two to three pairs of prominent tube feet, and sunken arm tips (Fig. 8 E). Stage VI : (R: 834 µm; N: 74). Broods still located in a mass outside the females' body. Fully developed arms, with four pairs of tube feet per arm, and aboral spines extending to arm tips (Fig. 8 F). In terms of temporality, stage I was observed during the austral autumn (from May to June), stage II prevailed in the early winter (June and early July). The stage III was detected between June and July, while stage IV was present in winter (from June to August). More advanced stages V and VI occurred during late winter to austral spring (August to September) (Table 1 ). 4. Discussion Our results showed that only 6.52% of females of Asterina fimbriata exhibited typical gametogenic stages associated with a periodic cycle while most of females exhibit an aperiodic reproductive pattern, as evidenced by the simultaneous presence of oocytes at different developmental stages within the gonads, and was no possible to identify a significant accumulation of mature oocytes. Although there was no correlation between oocytes diameter and environmental variables, two distinct peaks in mean oocyte size were detected in April-May. These peaks occurred after environmental variables had already begun to decline, and were followed by a decrease in oocyte size from June onward. This pattern suggests that a small proportion of females completes final maturation at the onset of the cold season and releases mature oocytes between May and August. Notably, these periods coincide with the presence of brooding females, indicating that those producing large oocytes are likely the same females that subsequently brood their offspring. The occasional increases in average oocytes diameter during the study period reflect reproductive activity restricted to a minority of females, rather than a synchronized spawning event at the population level. This interpretation is consistent with the overall stability in oocyte size distribution (particularly 50–150 µm range), which remained relatively constant throughout the year. Similar results have been found in other echinoderms (Giese and Pearse 1974 ; Gage and Tyler 1982 ; Brogger et al. 2013 ), and are commonly observed in species inhabiting warm or deep-sea environments. In deep-sea species, the lack of seasonality may result from the stable and low-temperature conditions typical of those habitats, where reproductive cycles are less influenced by environmental factors (Pain et al. 1982 ; Hendler 1991 ; Tyler and Young 1992 ; Young 2003 ; Mercier and Hamel 2009 ; Berecoechea et al. 2017 ; Rivadeneira et al. 2017 ). Similar findings have been reported for other sea stars species (Shick et al. 1981 ; Pearse et al. 1991 ; Byrne 1996 ; Benítez-Villalobos et al. 2007 ; Benítez-Villalobos and Díaz-Martínez 2010 ; Rivadeneira et al. 2017 ), as well as for other echinoderms (Byrne 1989 ; Brogger et al. 2013 ; Berecoechea et al. 2017 ; Martinez et al. 2018). Species such as Leptasterias tenera (Worley et al. 1977 ), Microphiopholis gracillima (Singletary 1980 ), and Ophioplocus januarii (Brogger et al. 2013 ) have been observed to spawn a limited number of oocytes into the water column. Based on the continuous presence of early-stage oocytes, the low abundance of mature oocytes, and the few females that spawn, we suggest that A. fimbriata follows a similar reproductive strategy. Although continuous reproduction appears to be the dominant pattern in Asterina fimbriata , males exhibit a distinct annual reproductive cycle, similar to observations in other Southern Hemisphere species, such as Anasterias minuta (Salvat 1985 ) and A. antarctica (Laptikhovsky et al. 2015 ), as well as Northern Hemisphere species, such as Leptasterias pusilla (Smith 1971 ). Furthermore, spermatogonia were observed throughout the study period, although they were more prominent following the spawning stage. Similarly, Pearse ( 1965 ) and Worley et al. ( 1977 ) reported the year-round presence of spermatogonia in Odontaster validus and Leptasterias tenera , respectively. Additionally, we observed that male A. fimbriata from Camarones Bay spawned their spermatozoa during austral winter between June and August, the coldest months in the region. The spawning period of males coincides with that of females. These events may be associated with declining seawater temperatures, as previously observed in other species, such as A. minuta (Gil and Zaixso 2007 ), L. littoralis (O’brien 1976 ), and N. georgianus (Bosch and Slattery 1999 ). Since phagocytes typically appear following spawning events (Ferrand 1983 ) and toward the end of the reproductive cycle (Worley et al. 1977 ), their presence in 40% of females throughout the study period suggests that the persistent occurrence of phagocytes in A. fimbriata may play a role in energy allocation for reproduction. This process could represent a mechanism of nutrient recycling within the ovary, as proposed by Smith ( 1971 ), whereby cytoplasmic components from phagocytized oocytes are subsequently transferred to developing oocytes. This observation may reflect an indirect role of phagocytes in oocyte nutrition, as suggested by Cognetti and Delavault ( 1962 ), who proposed that degradation products released during phagocytosis are assimilated by developing oocytes, thereby optimizing energy utilization. This energy recycling could be related to the low percentage of females spawning and to the feeding strategy of A. fimbriata Regarding brooding, this study demonstrates that the brooding period of A. fimbriata lasted approximately four months, from May to September, culminating in the release of juveniles. Broods of A. fimbriata completed their development during austral spring (September), when the temperature ranged 9.5ºC and environmental conditions become more favourable for survival. This pattern is typical of temperate regions, where brooding occurs during the colder months, followed by the release of juveniles in warmer months (Ferrand 1983 ; Clarke 1987 ; McClintock and Pearse 1987 ; Hamel and Mercier 1995 ; Gil and Zaixso 2007 ; Pérez et al. 2008 ). Asterina fimbriata exhibits a brooding period similar in duration to that of Anasterias minuta , another Patagonian species that broods from austral autumn (March–April) to spring (October–November) (Salvat 1985 ; Gil et al. 2011 ). A comparable brooding duration has also been observed in Bernasconiaster pipi , a species found in the deep waters of the Mar del Plata Submarine Canyon, although this likely occurs under more environmentally stable conditions (Rivadeneira et al. 2020 ). Both brooding and non-brooding females exhibited a continuous production of previtellogenic oocytes. This finding is noteworthy, as one would expect larger oocytes in non-brooding females, consistent with the findings of Gil and Zaixso ( 2007 ). These authors observed either previtellogenic oocytes or, in some cases, an absence of oocytes in the ovaries of brooding females, whereas in non-brooding females exhibited increased oocyte size and the presence of vitellogenic oocytes. Moreover, gonad size in females remained unchanged throughout the study period, which is unusual, as seasonal variations in gonad size are typically expected in non-brooding females (Chia and Walker 1991 ). Even among brooding females, no notable changes in external gonad morphology were detected during the brooding period. This observation aligns with the findings of Raymond et al. ( 2004 ) for Leptasterias polaris , where no significant changes in female gonad size were observed during brooding. In our study, the disappearance of large oocytes shortly before the appearance of brooding females, suggests that few females spawn after develop mature oocytes and must allocate energy to the brooding period. This interpretation is one of the most notable findings, as it makes straight with the consistently low proportion of brooding females in the population (8%). This reflects the high energetic costs associated with brooding, particularly considering that brooding females do not feed during this period, and that Asterina fimbriata feeds primarily on detritus, as claws and shells of dead crabs, and secondly, on digested material (Alarcón et al . in prep.). The combination of continuous early-stage oocytes, the low proportion of mature oocytes, and the small number of brooding females indicates that only a limited proportion of the population can allocate sufficient energy necessary to reproduce and brooding. A consistently low percentage of brooding females (8%) was observed throughout the study period. Similar low frequencies have been reported for Anasterias rupicola , where only 30 out of 2,800 females were identified as brooders (Blankley and Branch 1984 ), as well as for Anasterias antarctica (Laptikhovsky et al. 2015 ). The low percentage of brooders observed in this study may be attributed to several factors. One possible explanation could be the high reproductive cost for females, as they cease feeding during the 4 months brooding period (Alarcón et al. in prep.), investing energy not only in the continuous production of oocytes, but also in self-maintenance and brood care. This finding suggests that the A. fimbriata population may be particularly fragile and vulnerable, given the energetic demands associated with brooding. It has been well-documented that brooding species tend to be small in size. Authors such as Strathmann and Strathmann ( 1982 ) and Byrne ( 1996 ) proposed the “energy hypothesis” to explain this relationship. This hypothesis suggests that small-sized females are unable to produce large numbers of widely dispersed offspring due to insufficient energy reserves required to sustain a highly fecund, dispersive reproductive strategy. As a result, they adopt a brooding strategy, investing greater energy in fewer offspring to enhance survival chances. Asterina fimbriata can brood between 5 and 99 offspring, a range similar to that observed in Smilasterias multipara (Komatsu et al. 2006 ), Asterina phylactica (Emson and Crump 1979 ), and Leptasterias pusilla (Smith 1971 ). Asterina fimbriata appears to follow a brooding strategy similar to that described by Lieberkind ( 1920 ) for the gastric brooding species Leptasterias groenlandica , as well as L. tenera (Hendler and Franz 1982 ) and Smilasterias multipara (Komatsu et al. 2006 ). In this strategy, females retain their broods within the stomach, and once the broods undergo metamorphosis, they emerge from the female's mouth and aggregate into a mass outside of it, where they remain protected by the female's body. Consistent with the findings of Hendler and Franz ( 1982 ), it can be suggested that A. fimbriata exhibits a developmental progression from internal to external brooding, with broods retained in the stomach during early stages and later emerging onto the oral surface as they mature. All gastric brooders produce eggs approximately 1.0 mm in diameter (Komatsu et al. 2006 ). At the onset of development, the diameter of A. fimbriata eggs measured 0.93 mm, which is comparable to values reported for other brooding sea stars, such as S. multipara (1.0 mm) (Komatsu et al. 2006 ), L. groenlandica (0.8–1.0 mm) (Fisher 1930 ), L. tenera (0.9–1.0 mm) (Worley et al. 1977 )d hexactis (0.9 mm) (Chia 1966 ). The eggs of A. fimbriata were surrounded by a transparent membrane during the early developmental stages, consistent with the descriptions identified as a fertilization membrane according to Gil et al. ( 2011 ) and Rivadeneira et al. ( 2020 ). Although various modes of brooding have been documented within the Asterinidae, as reported by Strathmann et al. ( 1984 ), Chen and Chen ( 1992 ), Byrne ( 1996 ), and Byrne et al. ( 2003 ), gastric brooding had never been recorded, until now. This study establishes Asterina fimbriata as the first documented case of gastric brooding within this family, marking a significant advancement in the understanding of its reproductive biology. Beyond expanding knowledge of A. fimbriata ’s developmental mode, these findings highlight the remarkable diversity of reproductive strategies within echinoderms and underscore the evolutionary adaptations that enable survival in dynamic environments. As research advances, A. fimbriata emerges as a key model for understanding reproductive specialization, reinforcing the importance of continued exploration to uncover the mechanisms that shape life in the world’s oceans. Declarations Funding This work was partially supported by a Doctoral Fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and by PICT 2019-2549. A part of this project was supported by funds from The Explorers Club Foundation. Competing Interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ariana Belen Alarcon Saavedra reports financial support was provided by The Explorers Club Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contributions Alarcón Saavedra Ariana Belén, Bigatti Gregorio and Brogger Martín Ignacio contributed to the study conceptualization, design and resources. Methodology was performed by Alarcón Saavedra Ariana Belén and Bigatti Gregorio. Formal analysis was performed by Alarcón Saavedra Ariana Belén and Rebolledo Sol Aylén. Alarcón Saavedra Ariana Belén and Brogger Martín Ignacio contributed to funding acquisition. The first original draft of the manuscript was written by Alarcón Saavedra Ariana Belén, and all authors commented on subsequent versions of the manuscript. All authors read and approved the final manuscript. Data Availability The dataset used in this study can be made available upon request to the corresponding author. Ethics approval The study has all permits approved during the samplings. These permits from the National Parks Administration and the province of Chubut (Argentina) are necessary to enter the natural area, dive and collect samples in order to carry out the proposed research. Acknowledgements We would like to express our gratitude to the Park Rangers in Camarones Bay, Chubut for providing housing during field trips. We are also grateful to Pablo Sugliano, Cecilia Astengo, and Juan Pombo (Parque Interjurisdiccional Marino Costero Patagonia Austral - PIMCPA) for their assistance during dives. Special thanks to Julia Marcos for her help throughout the sample collection, as well as to other colleagues and technicians for their support during field trips. We also thank Marcelo Santos for histological assistance. Additionally, we extend our gratitude to the members of Laboratorio de Reproducción y Biología Integrativa de Invertebrados Marinos (LARBIM) for their constant support. 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In: Tyler PA (ed) Ecosystems of the deep oceans. Elsevier, Amsterdam, pp 381–426 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 07 Jan, 2026 Reviewers invited by journal 07 Jan, 2026 Editor assigned by journal 26 Dec, 2025 First submitted to journal 25 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8451161","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":571115713,"identity":"b8d70986-58e9-4d8d-9068-fb986575332b","order_by":0,"name":"Ariana Belén Alarcón 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07:10:39","extension":"html","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172879,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/6fc68655c46f918639396dac.html"},{"id":100362578,"identity":"030d0ba9-6b3f-4262-8410-fa20fc7efbee","added_by":"auto","created_at":"2026-01-16 07:47:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8931355,"visible":true,"origin":"","legend":"\u003cp\u003eOvaries of \u003cem\u003eAsterina fimbriata\u003c/em\u003e showing oocytes at different developmental stages: previtellogenic oocytes (po), intermediate oocytes (io), and vitellogenic oocytes (vo), along with the outer sac (os) and inner sac (is). (A) Ovaries containing phagocytes (arrowheads). (B, C) Ovaries showing signs of partial spawning, with empty spaces in the lumen (lu), numerous previtellogenic oocytes (po), and some intermediate (io) and vitellogenic oocytes (vo). (D) Ovaries containing vitellogenic oocytes (vo) and intermediate oocytes (io). Scale bars: A, B, D= 200 µm; C= 100 µm.\u003c/p\u003e","description":"","filename":"Figure1JPG.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/ad0b7f114ab4c299420d200b.png"},{"id":100017484,"identity":"b048869b-ee9b-405c-964d-85fdd87e3982","added_by":"auto","created_at":"2026-01-12 07:10:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":975498,"visible":true,"origin":"","legend":"\u003cp\u003eMonthly oocyte size-frequency histograms of \u003cem\u003eAsterina fimbriata\u003c/em\u003e from July 2021 to June 2023. Sampling was not conducted in February 2023 and May 2023 due to adverse environmental conditions. N, number of females; n, number of oocytes measured. Mainly spawning events (arrow) during austral winter months (June–August).\u003c/p\u003e","description":"","filename":"Figure2TIFF.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/682b4416379ec5d5c401d2c1.png"},{"id":100362124,"identity":"1fbb94fa-0317-46b4-b5ae-7614666b3c45","added_by":"auto","created_at":"2026-01-16 07:46:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22603386,"visible":true,"origin":"","legend":"\u003cp\u003eMonthly frequency of female gonadal developmental stages in \u003cem\u003eAsterina fimbriata\u003c/em\u003e from July 2021 to June 2023. Growing stages were predominant during all the study period, and spawning stages were predominant during austral winter months (May–September), when water temperatures reached their lowest values (8–9ºC).\u003c/p\u003e","description":"","filename":"Figure3TIFF.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/d770cdcf1ceb674384679cf5.png"},{"id":100362616,"identity":"5cdb5e9a-cf98-4b95-b059-bbb17e0d49c7","added_by":"auto","created_at":"2026-01-16 07:47:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":511593,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variation of seawater temperature (ºC), day length (hours), and average oocyte diameter (µm) in \u003cem\u003eAsterina fimbriata\u003c/em\u003e from July 2021 to June 2023. Temperature and day length are shown on a relative scale (left Y-axis), while oocyte diameter is plotted on the right Y-axis.\u003c/p\u003e","description":"","filename":"Figure4TIFF.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/fa0b36e5f642a85186b6036b.png"},{"id":100017490,"identity":"4db9211b-76c6-4603-8945-0f8453552ab9","added_by":"auto","created_at":"2026-01-12 07:10:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14519134,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular development stages of \u003cem\u003eAsterina fimbriata\u003c/em\u003e. (A) Growing stage, showing spermatozoa (sz), spermatic columns composed of spermatogonia (sg) and spermatocytes (sc), and the outer sac (os). (B) Mature stage, with testes filled with spermatozoa and shortened spermatic columns (sc), sometimes absent. (C) Spawning stage, characterized by few spermatogonia (sg), empty spaces, and relict spermatozoa in the lumen (lu). (D) Post-spawning stage, with spermatogonia (sg) predominantly in the lumen, along with the outer sac (os) and inner sac (is). (E) Resorption stage, showing phagocytes (ph) within the lumen. (F) Testes exhibiting two simultaneous developmental stages: mature regions with a mass of spermatozoa (sz) and spawning testes with empty spaces and relict spermatozoa in the lumen (lu). Scale bars: A, D, E= 20 µm; B, C, F= 100 µm.\u003c/p\u003e","description":"","filename":"Figure5JPG.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/233c3016cb0635d4502e6ff7.png"},{"id":100362614,"identity":"6d256078-cfae-47dd-917a-f1a08dc13294","added_by":"auto","created_at":"2026-01-16 07:47:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3204315,"visible":true,"origin":"","legend":"\u003cp\u003eMonthly frequency of male gonadal developmental stages in \u003cem\u003eAsterina fimbriata\u003c/em\u003e from July 2021 to June 2023. Spawning stages were predominant during the austral winter months (June–August), when water temperatures reached their lowest values (8–9ºC).\u003c/p\u003e","description":"","filename":"Figure6JPG.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/ebc2df73d9cd41eb53995e1f.png"},{"id":100362579,"identity":"6c1ea591-300c-4354-a70f-5b5eb1873a08","added_by":"auto","created_at":"2026-01-16 07:47:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6984793,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) Broods (arrowheads) located within the female's stomach. (C) Broods positioned near the mouth and partially visible externally. (D) Broods fully exposed outside the female’s mouth, forming a mass on the oral surface beneath the arched arms. Scale bars: A= 2 mm; B, C= 500 µm; D= 5 mm.\u003c/p\u003e","description":"","filename":"Figure7JPG.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/c4cf97b02b373d28c1e1ae47.png"},{"id":100362514,"identity":"28484c22-9950-4547-aca5-21b442eadd7b","added_by":"auto","created_at":"2026-01-16 07:46:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9741010,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent stages of development (A-F) of \u003cem\u003eAsterina fimbriata\u003c/em\u003e. (A–A') Earliest embryos stage located inside females'stomachs; tm: transparent membrane. (B-B') Oval embryos; s: sunken at opposite extremes; some embryos have one or two depressions in their body (black arrows). (C) Aboral view; incipient tube feet structures emerge through the body wall (arrowhead), defining the future formation of the ambulacral groove and the oral surface. (D) Oral view; broods began to peep out from the females' mouths; the body of broods adopted a slightly pentagonal shape; the future arms with two pairs of tube feet (tf). (E) Oral view; the broods were located in a mass outside the females' bodies, on the oral surface; the final form as adults begins to define in these broods. The ambulacral groove becomes distinguishable, and the centre of the future mouth becomes evident. The arms have two to three pairs of prominent tube feet (tf); there are sunken areas at the tip of each arm (white arrowhead); ag: Ambulacral groove, mo: Mouth. (F) Oral view; the arms are developed, each with about four pairs of tube feet (tf); the ambulacral groove was clearly distinguishable; ag: Ambulacral groove; the abactinal area is covered to spines (white arrowhead). Scale bars: A= 1 mm; A', B, E= 500 µm; B'= 400 µm; C, D, F= 200 µm.\u003c/p\u003e","description":"","filename":"Figure8JPG.png","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/bb3151b821a9ada7e3243ba6.png"},{"id":100381307,"identity":"a233465a-d1ce-4449-97fb-6df8d9f0ec01","added_by":"auto","created_at":"2026-01-16 10:38:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":71491717,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8451161/v1/6b224fa3-093f-4ce6-8f90-0e157e5fc22a.pdf"}],"financialInterests":"","formattedTitle":"First evidence of gastric brooding in Asterinidae highlighted by the reproductive strategy of Asterina fimbriata in Atlantic Patagonia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEchinoderms exhibit a wide diversity of reproductive strategies. In general, sea stars from cold and temperate waters display cyclical reproductive cycles, often characterized by annual or seasonal patterns (Mercier and Hamel \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Cossi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These cyclical reproductive patterns are closely linked to environmental factors, such as temperature and photoperiod, which fluctuate according to predictable seasonal regimes (Mercier and Hamel \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, some warm-water or deep-water species exhibit a continuous reproductive pattern associated with relatively stable environments, where environmental conditions remain largely unchanged (Byrne et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Ben\u0026iacute;tez-Villalobos et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mercier and Hamel \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rivadeneira et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Interestingly, seasonal reproductive patterns have also been reported in species traditionally considered to have continuous reproduction, including deep-waters species (Tyler et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and shallow-water species from cold environments (Brogger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Mercier and Hamel (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), along with other researchers, suggest that the term \u0026ldquo;aperiodic\u0026rdquo; is more appropriate than \u0026ldquo;continuous\u0026rdquo; when referring to the absence of a detectable reproductive pattern. In species with cyclic reproduction, particularly those with annual periodicity, gametogenesis typically follows a clear, synchronous, and orderly sequence of developmental stages, as documented in several species (Carvalho and Ventura \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Gil and Zaixso \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gil et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, species with an aperiodic reproductive pattern present greater challenges in identifying distinct developmental stages, as gametes at various stages of maturation are simultaneously present within the gonads (Byrne \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Brogger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fraysse et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSea stars exhibit diverse modes and strategies of reproduction, ranging from broadcast spawning to species that incubate and brood their offspring (Byrne et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Byrne \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Gillespie and McClintock \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The latter strategy is less common and has evolved independently within Asteroidea (Emlet \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Chia and Walker \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Lawrence and Herrera \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Brooding is typically found in certain families, such as Ctenodiscidae (Rivadeneira et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), Pterasteridae (Fraysse et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Asteriidae (P\u0026eacute;rez et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rivadeneira et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and Asterinidae (Byrne \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). This reproductive strategy appears to be characteristic of species inhabiting high and mid-latitudes (Mileikovsky \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Himmelman et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; McEdward \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Bosch and Slattery \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Fraysse et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, brooding strategies may occur within the parent\u0026rsquo;s body, such as inside the gonads, as observed in \u003cem\u003ePatiriella vivipara\u003c/em\u003e, \u003cem\u003eP. parvivipara\u003c/em\u003e, and \u003cem\u003eCrysptasterina hystera\u003c/em\u003e (Byrne \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), or inside the stomach, as seen in \u003cem\u003eSmilasterias multipara\u003c/em\u003e (O\u0026rsquo;Loughlin and O\u0026rsquo;Hara \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Other species possess specialized brood chambers located in different parts of the parent\u0026rsquo;s body, including the aboral and oral surfaces, as well as beneath the body. Examples include \u003cem\u003eCtenodiscus australis\u003c/em\u003e (Rivadeneira et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eDiplopteraster verrucosus\u003c/em\u003e (Fraysse et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eAnasterias minuta\u003c/em\u003e (Salvat \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Gil et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and \u003cem\u003eAnasterias antarctica\u003c/em\u003e (P\u0026eacute;rez et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring brooding, some females can continue feeding while incubating their offspring. For instance, individuals of \u003cem\u003eAnasterias rupicola\u003c/em\u003e have been observed feeding while brooding (Blankley and Branch \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Similarly, \u003cem\u003eLeptasterias littoralis\u003c/em\u003e can consume particulate material during this period (O\u0026rsquo;Brien \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Other species, such as \u003cem\u003eLeptasterias pusilla\u003c/em\u003e and \u003cem\u003eL. tenera\u003c/em\u003e, have been found with digested material in their mouths while brooding (Osterud \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1918\u003c/span\u003e; Smith \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Hendler and Franz \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). In contrast, some sea stars do not feed while brooding, including \u003cem\u003eAnasterias minuta\u003c/em\u003e (Gil et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), A. \u003cem\u003eantarctica\u003c/em\u003e (P\u0026eacute;rez et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and \u003cem\u003eLeptasterias hexactis\u003c/em\u003e (Chia \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Understanding whether brooding females feed or not is essential to assess the energetic investment associated with this reproductive strategy.\u003c/p\u003e \u003cp\u003eThe family Asterinidae exhibits a variety of life histories (Dartnall \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Byrne and Cerra \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Byrne \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and within the order Valvatida, brooding has been reported in several species (Bosch and Pearse \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Byrne \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pearse et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), particularly within the genera \u003cem\u003eAsterina\u003c/em\u003e, one of the richest genera in terms of reproductive strategies (Rowe and Gates \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The genus \u003cem\u003eAsterina\u003c/em\u003e comprises more than 30 species worldwide, of which only two, \u003cem\u003eA. stellifera\u003c/em\u003e and \u003cem\u003eA. fimbriata\u003c/em\u003e, have been reported in Argentina (Bernasconi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Meretta et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eAsterina stellifera\u003c/em\u003e is larger than \u003cem\u003eA. fimbriata\u003c/em\u003e and is distributed from Cabo Frio, Brazil (23\u0026deg;S, 42\u0026deg;W), to Mar del Plata, Argentina (38\u0026deg;S, 56\u0026deg;W), in the southwestern Atlantic Ocean (Clark and Downey \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). This species follows a synchronous reproductive cycle and releases its gametes into the water column (Carvalho and Ventura \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In contrast, \u003cem\u003eA. fimbriata\u003c/em\u003e is a smaller sea star with a moderately flattened, pentagonal body, displaying limited differentiation between its arms and central disc (Bernasconi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). This species has been recorded throughout southern Patagonia (Argentina and Chile), from approximately 41\u0026deg; S southward to the Magellanic region, including Camarones Bay (Chubut, Argentina), the Beagle Channel, and \u003cem\u003eMacrocystis\u003c/em\u003e kelp forests located south of the Strait of Magellan. It is present in both the Atlantic and Pacific sectors and occurs from intertidal zones down to depths of nearly 300 meters, inhabiting various substrates such as rock, gravel, sand, mud, and kelp (Bernasconi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Clark and Downey \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Currently, knowledge regarding the biology of this species remains scarce.\u003c/p\u003e \u003cp\u003eThe present study investigates the reproductive biology of \u003cem\u003eAsterina fimbriata\u003c/em\u003e through monthly histological analyses of the gonads and by describing its brooding behaviour. This includes the characterization of offspring developmental stages, examination changes in brood size, and identification of morphological variations. Additionally, the study discusses the reproductive strategy of brooding in relation to established patterns observed in other sea star species, highlighting the associated advantages and disadvantages in terms of energy investment and feeding behaviour during the brooding period.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study area and field sampling\u003c/h2\u003e \u003cp\u003eBetween 20 and 30 specimens of \u003cem\u003eAsterina fimbriata\u003c/em\u003e were collected monthly by SCUBA diving at depths of 4 to 8 m in Camarones Bay, Chubut, Argentina (44\u0026deg;46'S, 65\u0026deg;34'W), from July 2021 to June 2023. Most specimens had a radius greater than 5 mm, though a few smaller individuals were found on hard rocky substrates covered with various algae and kelp forests, primarily \u003cem\u003eUndaria pinnatifida\u003c/em\u003e and \u003cem\u003eMacrocystis pyrifera\u003c/em\u003e. \u003cem\u003eAsterina fimbriata\u003c/em\u003e typically inhabits numerous cavities and crevices within the rocks, which provide shelter. This locality is a bay with open waters to the sea (Schillizzi et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with an attenuation coefficient (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e) ranging from 0.25 to 0.31 m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Villafa\u0026ntilde;e et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the field, all specimens were fixed in Bouin\u0026rsquo;s solution for 48 hours and subsequently transferred to 70% ethanol. Each specimen was measured using a Vernier calliper to determine the radius (R), defined as the distance from the centre of the disc to the tip of the longest arm, and the radius (r), defined as the distance from the centre of the disc to the interradius. The gonads were then dissected and preserved for histological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sex determination and histological analysis\u003c/h2\u003e \u003cp\u003eFor histological analysis, the gonads were dehydrated in graded ethanol solutions and embedded in paraffin. Sections of 5\u0026ndash;6 \u0026micro;m thickness were cut and stained with haematoxylin and eosin. The diameter of each oocyte, with a distinct nucleolus, was measured as the mean value between the maximum diameter and its perpendicular at the oocyte\u0026rsquo;s centre (McClary and Mladenov \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Byrne \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Bosch and Slattery \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Measurements were performed using AxioVision SE64, Rel. 4.9.1 software, and gonad sections were photographed under a Stemi 2000-C stereomicroscope and a ZEISS Axiocam ERc5s camera.\u003c/p\u003e \u003cp\u003eFor sex determination, gonads were examined microscopically, and sex was confirmed through histological analysis. A Chi-squared test was applied to assess significant differences in sex ratios throughout the study period and for each month individually.\u003c/p\u003e \u003cp\u003eThe seawater temperature and depth were recorded using a dive computer. The pH was measured using a \u0026ldquo;Milwaukee\u0026rdquo; pH meter, and salinity was measured with a field ATC refractometer. All parameters were recorded monthly in situ in each sampling. The monthly daylight average (photoperiod) was acquired from the Servicio de Hidrograf\u0026iacute;a Naval (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We related the oocyte diameter measurements from cells with a distinct nucleolus in all females with environmental parameters to estimate the gonadal cycle as was done by Nieto Vilela et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Cumplido et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Brooding and developmental stages\u003c/h2\u003e \u003cp\u003eAll specimens were thoroughly examined to identify different stages of brood development inside the parent\u0026rsquo;s body and oral cavity, using a stereoscopic magnifier. Offspring were counted, measured, and photographed. Various terms have been used in the literature to denote incubating parents, embryos, young, juveniles, and offspring (see: (Byrne et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Gil et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Byrne \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rivadeneira et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Fraysse et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the term \u0026ldquo;broods\u0026rdquo; refers to offspring produced and cared for by a single parent, while \u0026ldquo;brooding females\u0026rdquo; strictly refers to females exhibiting brooding behaviour.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sex determination\u003c/h2\u003e \u003cp\u003eOf the 572 individuals of \u003cem\u003eAsterina fimbriata\u003c/em\u003e studied, 276 were females, 292 were males, and 4 were immature. The sex ratio of the population did not differ significantly from 1:1 (χ\u0026sup2; = 0.47, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), nor did it differ significantly for each month (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The R ratio in the \u003cem\u003eA. fimbriata\u003c/em\u003e population ranged from 4.15 to 14.86 mm (mean\u0026thinsp;=\u0026thinsp;8.97 mm, SD\u0026thinsp;=\u0026thinsp;1.46 mm), while the r ratio varied from 2.98 to 10.12 mm (mean\u0026thinsp;=\u0026thinsp;6.40 mm, SD\u0026thinsp;=\u0026thinsp;1.04 mm). We found that the four smallest individuals (R\u0026thinsp;\u0026lt;\u0026thinsp;5 mm) lacked gonads or developing gametes and were therefore excluded from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Histological analyses\u003c/h2\u003e \u003cp\u003eHistological analysis was conducted for 260 females and 279 males. Each individual presented five pairs of gonads, located in the interradius on the aboral side of each arm. Ovaries were spherical and orange/yellow, while testes were branched with tubular structures and white in colour. The gonad morphology comprised two sacs: an outer sac of connective tissue and an inner sac of germinal cells, separated by a basal membrane. No notable changes in gonad external morphology were observed in females throughout the study period.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Females\u003c/h2\u003e \u003cp\u003eOocytes were classified into the following categories: \u003cb\u003eprevitellogenic oocytes\u003c/b\u003e, small, with thick walls, a distinguishable nucleolus, and a large nucleus; \u003cb\u003eintermediate oocytes\u003c/b\u003e: characterized by the presence of some yolk granules and thinner walls; and \u003cb\u003emature oocytes\u003c/b\u003e: filled with granules and lipid droplets, with thin walls. All females, including both brooders and non-brooders, exhibited ovaries containing oocytes at various developmental stages simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Previtellogenic (\u0026lt;\u0026thinsp;100 \u0026micro;m) and intermediate (100\u0026ndash;400 \u0026micro;m) oocytes were the most abundant across all months.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe identified structures compatible with certain gonadal stages in females each month through histological analysis. Phagocytes were observed in 40% of females throughout the study period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Developing ovaries containing oogonia, previtellogenic, and intermediate oocytes were also observed, although a pattern was not consistently observed across months. Only 18 females sampled had partially spawned ovaries during the periods from July to September 2021 and from November to December 2021, as well as from May to September 2022, from November to December 2022, and in June 2023. These ovaries were characterized by empty spaces in the lumen and the presence of some vitellogenic and previtellogenic oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, D). Accumulation of mature oocytes (\u0026gt;\u0026thinsp;400 \u0026micro;m) was detected only in 17 females (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.1 Oocytes size frequency distribution\u003c/h2\u003e \u003cp\u003eWe measured 6,011 oocytes with a distinct nucleolus, registering an average of 6 to 64 oocytes per female. The minimum oocyte diameter recorded was 17.82 \u0026micro;m (September 2021), while the maximum diameter was 916.31 \u0026micro;m (April 2023). Frequency histograms showed that the most common oocyte sizes ranged from 50 to 200 \u0026micro;m, indicating a constant presence of early-stage oocytes throughout all months (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although no significant accumulation of large oocytes (550\u0026ndash;600 \u0026micro;m) was detected, histograms showed a low frequency (\u0026lt;\u0026thinsp;10%) of these oocytes during the study period. The constant presence of early-stage oocytes throughout all months corresponded with the growing stage observed in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, the occurrence of large oocytes coincided with the presence of mature stage in some females, while their subsequent disappearance accorded with periods in which spawning females were recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to discover the influence of environmental variables in females\u0026rsquo; gonadal cycle, we noted that water temperature and day length followed a clear annual cycle, increasing during austral summer (December-February) and decreasing during austral winter (June-August) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The temperature reached a maximum of 17.5\u0026ordm;C in February at 4.5 meters depth, while day length reached a maximum of 15.53 h of light in December (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both variables were positively correlated (Pearson\u0026thinsp;=\u0026thinsp;0.76). Oocyte diameter showed weak fluctuations throughout the study period, with no strong seasonal trend. In addition, there was no correlation between oocyte diameter and day length (Pearson\u0026thinsp;=\u0026thinsp;0.01) or temperature (Pearson\u0026thinsp;=\u0026thinsp;0.19). However, in both 2022 and 2023, a mainly peak in mean oocyte diameter (\u0026gt;\u0026thinsp;250\u0026ndash;300 \u0026micro;m) was observed during April \u0026ndash;May, once environmental variables had already begun to decline. These peaks were followed by a decrease from June, indicating release of mature oocytes during the austral autumn\u0026ndash;winter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Males\u003c/h2\u003e \u003cp\u003eIn contrast to females, defined gonadal development stages were identifiable in males through histological analysis. Testes were classified into the following stages: 1) \u003cb\u003eGrowing stage\u003c/b\u003e: characterized by a spermatogonia layer resting on the inner sac, spermatic columns composed of spermatocytes, and some spermatozoa in the lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). 2) \u003cb\u003eMature stage\u003c/b\u003e: testes had slim walls, a thin spermatogonia layer, shorter spermatic columns, and a lumen filled with spermatozoa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). 3) \u003cb\u003eSpawning stage\u003c/b\u003e: testes exhibited thick walls, few spermatogonia, empty spaces, and residual spermatozoa in the lumen, occasionally accompanied by phagocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). 4) \u003cb\u003ePost-spawned stages\u003c/b\u003e: characterized by the presence of spermatogonia predominantly in the lumen, occasionally accompanied by phagocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). 5) \u003cb\u003eResorption stage\u003c/b\u003e: marked by phagocytes predominantly in the lumen, occasionally alongside spermatogonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). We noted that spermatogonia were present throughout the study period, even in mature males, becoming more evident during the post-spawning stages. Moreover, testes contained multiple developmental stages simultaneously, and mature and spawning testes were occasionally observed within the same individual (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.1 Temporal trend of testicular development\u003c/h2\u003e \u003cp\u003eThe temporal analysis of testicular development indicated an annual gametogenic cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The growing stage was predominantly observed during the austral summer (December and January), when temperatures ranged 15\u0026ndash;16\u0026ordm;C. The mature stage occurred mainly from the austral summer to mid-autumn (February to May, including June 2023), when temperatures ranged 17\u0026ndash;10\u0026ordm;C. The spawning stage was predominantly observed during the austral winter (from June to August), when temperatures ranged approximately 10\u0026ndash;8\u0026ordm;C. The post-spawning stage was mainly observed during austral spring (from September to November), when temperatures ranged 9\u0026ndash;11\u0026ordm;C. Finally, testes in resorption stage were observed throughout the year, with a higher incidence (10%\u0026ndash;30%) from March to November 2022.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Brooding behaviour and developmental stages\u003c/h2\u003e \u003cp\u003eWe studied three brooding seasons from July 2021 to June 2023, identifying 2 to 5 brooding females per month between May and September, totalling 23 brooding females, representing 8% of the total females studied. The R ratio in brooders ranged from 8 to 12.7 mm (mean\u0026thinsp;=\u0026thinsp;9.48 mm, SD\u0026thinsp;=\u0026thinsp;1.42 mm), and we recorded 505 broods, with 5 to 99 broods per female (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Most broods were located in a mass outside the female\u0026rsquo;s mouth, although some were found inside the females.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDevelopmental stages of broods recorded in \u003cem\u003eAsterina fimbriata\u003c/em\u003e from July 2021 to June 2023. The table shows the number of broods per female and the corresponding developmental stages observed in each brooding female per sampling month.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of broods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDevelopmental stages observed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e07/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eII-III-IV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e07/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIII-IV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e08/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIV-V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e08/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIV-V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV-VI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2022\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI-II\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e07/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIV-V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV-VI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIV-V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eII-IV-V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV-VI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2023\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI-II\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eII-III-IV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI-II\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEarly developmental stages were located in the stomach (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B), intermediate stages were found near the mouth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), and fully developed broods were observed in a mass outside the mouth, beneath the females' arched arms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Although a general spatial pattern of brood development was observed, with broods progressing from the stomach toward the mouth as development advanced, this pattern was not always strictly followed. In some females, broods at different developmental stages were found co-occurring within the same anatomical region, including more advanced stages still in the stomach or earlier stages near the mouth (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We identified six developmental stages, classified by size and morphological characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStage I\u003c/b\u003e: (Mean diameter: 929 \u0026micro;m; N: 121). Broods located in the stomachs of females. They were spherical or oval, bright yellow, surrounded by a transparent membrane, with no visible structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStage II\u003c/b\u003e: (Mean diameter: 1154 \u0026micro;m; N: 104). Broods still located in the females' stomachs, but they adopted an oval and elongated shape, surrounded by a transparent membrane. Some broods exhibited a depression in the middle of the body, while others displayed two depressions at opposite extremes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStage III\u003c/b\u003e: (Mean diameter: 1146 \u0026micro;m; N: 48). Broods still located in the females' stomachs. Incipient tube feet emerged through the body wall, indicating the future formation of the ambulacral groove and the oral surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStage IV\u003c/b\u003e: (Mean diameter: 1174 \u0026micro;m; N: 68). Broods peeping from the females' mouths, visible externally. Body of broods with pentagonal shape, with two pairs of tube feet per radius (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStage V\u003c/b\u003e: (R: 756 \u0026micro;m; N: 90). Broods located in a mass outside the females' body on the oral surface. Adult-like shape, with ambulacral grooves distinguishable, two to three pairs of prominent tube feet, and sunken arm tips (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStage VI\u003c/b\u003e: (R: 834 \u0026micro;m; N: 74). Broods still located in a mass outside the females' body. Fully developed arms, with four pairs of tube feet per arm, and aboral spines extending to arm tips (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eIn terms of temporality, stage I was observed during the austral autumn (from May to June), stage II prevailed in the early winter (June and early July). The stage III was detected between June and July, while stage IV was present in winter (from June to August). More advanced stages V and VI occurred during late winter to austral spring (August to September) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur results showed that only 6.52% of females of \u003cem\u003eAsterina fimbriata\u003c/em\u003e exhibited typical gametogenic stages associated with a periodic cycle while most of females exhibit an aperiodic reproductive pattern, as evidenced by the simultaneous presence of oocytes at different developmental stages within the gonads, and was no possible to identify a significant accumulation of mature oocytes.\u003c/p\u003e \u003cp\u003eAlthough there was no correlation between oocytes diameter and environmental variables, two distinct peaks in mean oocyte size were detected in April-May. These peaks occurred after environmental variables had already begun to decline, and were followed by a decrease in oocyte size from June onward. This pattern suggests that a small proportion of females completes final maturation at the onset of the cold season and releases mature oocytes between May and August. Notably, these periods coincide with the presence of brooding females, indicating that those producing large oocytes are likely the same females that subsequently brood their offspring.\u003c/p\u003e \u003cp\u003eThe occasional increases in average oocytes diameter during the study period reflect reproductive activity restricted to a minority of females, rather than a synchronized spawning event at the population level. This interpretation is consistent with the overall stability in oocyte size distribution (particularly 50\u0026ndash;150 \u0026micro;m range), which remained relatively constant throughout the year. Similar results have been found in other echinoderms (Giese and Pearse \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Gage and Tyler \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Brogger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and are commonly observed in species inhabiting warm or deep-sea environments. In deep-sea species, the lack of seasonality may result from the stable and low-temperature conditions typical of those habitats, where reproductive cycles are less influenced by environmental factors (Pain et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Hendler \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Tyler and Young \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Young \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Mercier and Hamel \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Berecoechea et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rivadeneira et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similar findings have been reported for other sea stars species (Shick et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Pearse et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Byrne \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Ben\u0026iacute;tez-Villalobos et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ben\u0026iacute;tez-Villalobos and D\u0026iacute;az-Mart\u0026iacute;nez \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rivadeneira et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as well as for other echinoderms (Byrne \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Brogger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Berecoechea et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Martinez et al. 2018). Species such as \u003cem\u003eLeptasterias tenera\u003c/em\u003e (Worley et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), \u003cem\u003eMicrophiopholis gracillima\u003c/em\u003e (Singletary \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), and \u003cem\u003eOphioplocus januarii\u003c/em\u003e (Brogger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) have been observed to spawn a limited number of oocytes into the water column. Based on the continuous presence of early-stage oocytes, the low abundance of mature oocytes, and the few females that spawn, we suggest that \u003cem\u003eA. fimbriata\u003c/em\u003e follows a similar reproductive strategy.\u003c/p\u003e \u003cp\u003eAlthough continuous reproduction appears to be the dominant pattern in \u003cem\u003eAsterina fimbriata\u003c/em\u003e, males exhibit a distinct annual reproductive cycle, similar to observations in other Southern Hemisphere species, such as \u003cem\u003eAnasterias minuta\u003c/em\u003e (Salvat \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) and \u003cem\u003eA. antarctica\u003c/em\u003e (Laptikhovsky et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), as well as Northern Hemisphere species, such as \u003cem\u003eLeptasterias pusilla\u003c/em\u003e (Smith \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Furthermore, spermatogonia were observed throughout the study period, although they were more prominent following the spawning stage. Similarly, Pearse (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1965\u003c/span\u003e) and Worley et al. (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) reported the year-round presence of spermatogonia in \u003cem\u003eOdontaster validus\u003c/em\u003e and \u003cem\u003eLeptasterias tenera\u003c/em\u003e, respectively. Additionally, we observed that male \u003cem\u003eA. fimbriata\u003c/em\u003e from Camarones Bay spawned their spermatozoa during austral winter between June and August, the coldest months in the region. The spawning period of males coincides with that of females. These events may be associated with declining seawater temperatures, as previously observed in other species, such as \u003cem\u003eA. minuta\u003c/em\u003e (Gil and Zaixso \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), \u003cem\u003eL. littoralis\u003c/em\u003e (O\u0026rsquo;brien \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), and \u003cem\u003eN. georgianus\u003c/em\u003e (Bosch and Slattery \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince phagocytes typically appear following spawning events (Ferrand \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and toward the end of the reproductive cycle (Worley et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), their presence in 40% of females throughout the study period suggests that the persistent occurrence of phagocytes in \u003cem\u003eA. fimbriata\u003c/em\u003e may play a role in energy allocation for reproduction. This process could represent a mechanism of nutrient recycling within the ovary, as proposed by Smith (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1971\u003c/span\u003e), whereby cytoplasmic components from phagocytized oocytes are subsequently transferred to developing oocytes. This observation may reflect an indirect role of phagocytes in oocyte nutrition, as suggested by Cognetti and Delavault (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1962\u003c/span\u003e), who proposed that degradation products released during phagocytosis are assimilated by developing oocytes, thereby optimizing energy utilization. This energy recycling could be related to the low percentage of females spawning and to the feeding strategy of \u003cem\u003eA. fimbriata\u003c/em\u003e\u003c/p\u003e \u003cp\u003eRegarding brooding, this study demonstrates that the brooding period of \u003cem\u003eA. fimbriata\u003c/em\u003e lasted approximately four months, from May to September, culminating in the release of juveniles. Broods of \u003cem\u003eA. fimbriata\u003c/em\u003e completed their development during austral spring (September), when the temperature ranged 9.5\u0026ordm;C and environmental conditions become more favourable for survival. This pattern is typical of temperate regions, where brooding occurs during the colder months, followed by the release of juveniles in warmer months (Ferrand \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Clarke \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; McClintock and Pearse \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Hamel and Mercier \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Gil and Zaixso \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; P\u0026eacute;rez et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003eAsterina fimbriata\u003c/em\u003e exhibits a brooding period similar in duration to that of \u003cem\u003eAnasterias minuta\u003c/em\u003e, another Patagonian species that broods from austral autumn (March\u0026ndash;April) to spring (October\u0026ndash;November) (Salvat \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Gil et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A comparable brooding duration has also been observed in \u003cem\u003eBernasconiaster pipi\u003c/em\u003e, a species found in the deep waters of the Mar del Plata Submarine Canyon, although this likely occurs under more environmentally stable conditions (Rivadeneira et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth brooding and non-brooding females exhibited a continuous production of previtellogenic oocytes. This finding is noteworthy, as one would expect larger oocytes in non-brooding females, consistent with the findings of Gil and Zaixso (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These authors observed either previtellogenic oocytes or, in some cases, an absence of oocytes in the ovaries of brooding females, whereas in non-brooding females exhibited increased oocyte size and the presence of vitellogenic oocytes. Moreover, gonad size in females remained unchanged throughout the study period, which is unusual, as seasonal variations in gonad size are typically expected in non-brooding females (Chia and Walker \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Even among brooding females, no notable changes in external gonad morphology were detected during the brooding period. This observation aligns with the findings of Raymond et al. (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) for \u003cem\u003eLeptasterias polaris\u003c/em\u003e, where no significant changes in female gonad size were observed during brooding.\u003c/p\u003e \u003cp\u003eIn our study, the disappearance of large oocytes shortly before the appearance of brooding females, suggests that few females spawn after develop mature oocytes and must allocate energy to the brooding period. This interpretation is one of the most notable findings, as it makes straight with the consistently low proportion of brooding females in the population (8%). This reflects the high energetic costs associated with brooding, particularly considering that brooding females do not feed during this period, and that \u003cem\u003eAsterina fimbriata\u003c/em\u003e feeds primarily on detritus, as claws and shells of dead crabs, and secondly, on digested material (Alarc\u0026oacute;n \u003cem\u003eet al\u003c/em\u003e. in prep.). The combination of continuous early-stage oocytes, the low proportion of mature oocytes, and the small number of brooding females indicates that only a limited proportion of the population can allocate sufficient energy necessary to reproduce and brooding.\u003c/p\u003e \u003cp\u003eA consistently low percentage of brooding females (8%) was observed throughout the study period. Similar low frequencies have been reported for \u003cem\u003eAnasterias rupicola\u003c/em\u003e, where only 30 out of 2,800 females were identified as brooders (Blankley and Branch \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), as well as for \u003cem\u003eAnasterias antarctica\u003c/em\u003e (Laptikhovsky et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The low percentage of brooders observed in this study may be attributed to several factors. One possible explanation could be the high reproductive cost for females, as they cease feeding during the 4 months brooding period (Alarc\u0026oacute;n \u003cem\u003eet al.\u003c/em\u003e in prep.), investing energy not only in the continuous production of oocytes, but also in self-maintenance and brood care. This finding suggests that the \u003cem\u003eA. fimbriata\u003c/em\u003e population may be particularly fragile and vulnerable, given the energetic demands associated with brooding.\u003c/p\u003e \u003cp\u003eIt has been well-documented that brooding species tend to be small in size. Authors such as Strathmann and Strathmann (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) and Byrne (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) proposed the \u0026ldquo;energy hypothesis\u0026rdquo; to explain this relationship. This hypothesis suggests that small-sized females are unable to produce large numbers of widely dispersed offspring due to insufficient energy reserves required to sustain a highly fecund, dispersive reproductive strategy. As a result, they adopt a brooding strategy, investing greater energy in fewer offspring to enhance survival chances. \u003cem\u003eAsterina fimbriata\u003c/em\u003e can brood between 5 and 99 offspring, a range similar to that observed in \u003cem\u003eSmilasterias multipara\u003c/em\u003e (Komatsu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), \u003cem\u003eAsterina phylactica\u003c/em\u003e (Emson and Crump \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), and \u003cem\u003eLeptasterias pusilla\u003c/em\u003e (Smith \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAsterina fimbriata\u003c/em\u003e appears to follow a brooding strategy similar to that described by Lieberkind (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1920\u003c/span\u003e) for the gastric brooding species \u003cem\u003eLeptasterias groenlandica\u003c/em\u003e, as well as \u003cem\u003eL. tenera\u003c/em\u003e (Hendler and Franz \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) and \u003cem\u003eSmilasterias multipara\u003c/em\u003e (Komatsu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In this strategy, females retain their broods within the stomach, and once the broods undergo metamorphosis, they emerge from the female's mouth and aggregate into a mass outside of it, where they remain protected by the female's body. Consistent with the findings of Hendler and Franz (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), it can be suggested that \u003cem\u003eA. fimbriata\u003c/em\u003e exhibits a developmental progression from internal to external brooding, with broods retained in the stomach during early stages and later emerging onto the oral surface as they mature.\u003c/p\u003e \u003cp\u003eAll gastric brooders produce eggs approximately 1.0 mm in diameter (Komatsu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). At the onset of development, the diameter of \u003cem\u003eA. fimbriata\u003c/em\u003e eggs measured 0.93 mm, which is comparable to values reported for other brooding sea stars, such as \u003cem\u003eS. multipara\u003c/em\u003e (1.0 mm) (Komatsu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), L. \u003cem\u003egroenlandica\u003c/em\u003e (0.8\u0026ndash;1.0 mm) (Fisher \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1930\u003c/span\u003e), \u003cem\u003eL. tenera\u003c/em\u003e (0.9\u0026ndash;1.0 mm) (Worley et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1977\u003c/span\u003e)d \u003cem\u003ehexactis\u003c/em\u003e (0.9 mm) (Chia \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). The eggs of \u003cem\u003eA. fimbriata\u003c/em\u003e were surrounded by a transparent membrane during the early developmental stages, consistent with the descriptions identified as a fertilization membrane according to Gil et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Rivadeneira et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough various modes of brooding have been documented within the Asterinidae, as reported by Strathmann et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), Chen and Chen (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), Byrne (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), and Byrne et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), gastric brooding had never been recorded, until now. This study establishes \u003cem\u003eAsterina fimbriata\u003c/em\u003e as the first documented case of gastric brooding within this family, marking a significant advancement in the understanding of its reproductive biology. Beyond expanding knowledge of \u003cem\u003eA. fimbriata\u003c/em\u003e\u0026rsquo;s developmental mode, these findings highlight the remarkable diversity of reproductive strategies within echinoderms and underscore the evolutionary adaptations that enable survival in dynamic environments. As research advances, \u003cem\u003eA. fimbriata\u003c/em\u003e emerges as a key model for understanding reproductive specialization, reinforcing the importance of continued exploration to uncover the mechanisms that shape life in the world\u0026rsquo;s oceans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by a Doctoral Fellowship from the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET) and by PICT 2019-2549. A part of this project was supported by funds from The Explorers Club Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ariana Belen Alarcon Saavedra reports financial support was provided by The Explorers Club Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlarc\u0026oacute;n Saavedra Ariana Bel\u0026eacute;n, Bigatti Gregorio and Brogger Mart\u0026iacute;n Ignacio\u0026nbsp;contributed to the study conceptualization, design and resources.\u0026nbsp;Methodology was\u0026nbsp;performed by\u0026nbsp;Alarc\u0026oacute;n Saavedra Ariana Bel\u0026eacute;n and Bigatti Gregorio.\u0026nbsp;Formal analysis was\u0026nbsp;performed by\u0026nbsp;Alarc\u0026oacute;n Saavedra Ariana Bel\u0026eacute;n and Rebolledo Sol Ayl\u0026eacute;n. Alarc\u0026oacute;n Saavedra Ariana Bel\u0026eacute;n and\u0026nbsp;Brogger Mart\u0026iacute;n Ignacio contributed to funding acquisition.\u0026nbsp;The first original draft of the manuscript was written by\u0026nbsp;Alarc\u0026oacute;n Saavedra Ariana Bel\u0026eacute;n,\u0026nbsp;and all authors commented on subsequent versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset used in this study can be made available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has all permits approved during the samplings. These permits from the National Parks Administration and the province of Chubut (Argentina) are necessary to enter the natural area, dive and collect samples in order to carry out the proposed research.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to the Park Rangers in Camarones Bay, Chubut for providing housing during field trips. We are also grateful to Pablo Sugliano, Cecilia Astengo, and Juan Pombo (Parque Interjurisdiccional Marino Costero Patagonia Austral - PIMCPA) for their assistance during dives. Special thanks to Julia Marcos for her help throughout the sample collection, as well as to other colleagues and technicians for their support during field trips. We also thank Marcelo Santos for histological assistance. Additionally, we extend our gratitude to the members of Laboratorio de Reproducci\u0026oacute;n y Biolog\u0026iacute;a Integrativa de Invertebrados Marinos (LARBIM) for their constant support. This work was partially supported by a Doctoral Fellowship from the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET) and by PICT 2019-2549. A part of this project was supported by funds from The Explorers Club Foundation. This is contribution N\u0026ordm; xxx of the Laboratorio de Reproducci\u0026oacute;n y Biolog\u0026iacute;a Integrativa de Invertebrados Marinos (LARBIM).\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBernasconi I (1973) Asteroideos argentinos VI. Familia Asterinidae. 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Elsevier, Amsterdam, pp 381\u0026ndash;426\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sea stars, South Atlantic Ocean, Reproduction, Development","lastPublishedDoi":"10.21203/rs.3.rs-8451161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8451161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe reproductive biology of \u003cem\u003eAsterina fimbriata\u003c/em\u003e, a member of the family Asterinidae, was investigated through monthly sampling between July 2021 and June 2023 in Atlantic Patagonian coastal waters. This species presented a curious reproduction strategy: females exhibited an aperiodic reproductive pattern, characterized by the continuous presence of oocytes at different developmental stages co-occurring within the gonads and a low frequency of mature oocytes along the year. However, 6.52% of females exhibited a periodic oogenic cycle, showing a release maximum peak between May and September (coldest water temperature) Males displayed a clear annual gametogenic cycle, with maximum sperm release during the coldest months (June-August). Only 8% of females exhibiting brooding behaviour, which lasted approximately four months (May-September) and coincides after the major oocyte release season. Broods developed within the stomach and, following metamorphosis, emerged as a mass outside the female's mouth. Six distinct developmental stages were identified, revealing a progressive transition from internal to external brooding as offspring matured. \u003cem\u003eA. fimbriata\u003c/em\u003e represents the first confirmed case of gastric brooding in the family Asterinidae. The low proportion of reproductive females would be indicating a high energetic cost of incubation that could be associated to the feeding behaviour of this fragile species. These findings expand the understanding of reproductive strategies in sea stars and highlight a novel adaptation within this clade.\u003c/p\u003e","manuscriptTitle":"First evidence of gastric brooding in Asterinidae highlighted by the reproductive strategy of Asterina fimbriata in Atlantic Patagonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 07:10:30","doi":"10.21203/rs.3.rs-8451161/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-07T23:11:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T23:09:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-26T13:38:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biology","date":"2025-12-25T18:10:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ae80e72f-88b0-4504-8b77-833c12e22a4f","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-25T12:13:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 07:10:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8451161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8451161","identity":"rs-8451161","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}