Broodstock Conditioning of the Yellow Clam (Amarilladesma mactroides)

Abstract

Broodstock conditioning involves maintaining adult animals in optimal environmental conditions to ensure that the largest number of breeders reach maturity. We evaluated the gonadal development and the occurrence of spawns in the yellow clam (Amarilladesma mactroides) and estimated the duration of conditioning that results in the highest maturity rate. Clams were kept buried in a 10 cm sand bed within Ø = 15 cm containers. A concentrate of Isochrysis galbana and Chaetoceros muelleri was supplied daily. Four conditioning periods (14, 28, 45 and 60 days) with four replicates were used. On the day of collection and at the end of each conditioning period, clams were induced to spawn with thermal shocks (16.7 °C to 26 °C) and sperm. The first spawns were observed on day 28, but only 50% of the males and 33% of the females were mature, and their mean oocyte diameter (36.33 µm) was smaller than the minimum (45–50 µm) considered for ready-to-spawn clams. However, on day 45, all males and females were mature (mean oocyte diameter = 45.14 µm) and 1102 × 10³ eggs were released. To ensure a high maturity rate, yellow clams should be conditioned in captivity for 45 days. Monitoring gonadal development in the wild to collect clams at advanced stages of gonadal development may be a less expensive strategy worth considering.

1. Introduction

The yellow clam, Amarilladesma mactroides, is an ecologically and economically important bivalve naturally found on the Atlantic coast of South America. It is considered a temperate species as its geographical distribution range spans from Isla del Jabali, Province of Buenos Aires, Argentina, to Ilha Grande, Rio de Janeiro, Brazil [1,2]. In the intertidal zone of these beaches, the yellow clam is one of the main elements of the community structure [3,4] and is usually the mollusk with the highest biomass [5,6]. Given this abundance, A. mactroides has been collected for human consumption for at least 4000 years [7,8] and, more recently, to be used as bait for sport fishing [9,10]. While its capture and commercialization sustain traditional fishing communities in southern Brazil and Uruguay and are part of the regional food culture, there is no data on the volume captured annually.

Over the past three decades, several populations of A. mactroides have suffered episodes of mass mortality throughout their geographic range [2,4,11]. Although the causes of mass mortality events in natural populations are very difficult to ascertain, they are probably due to a combination of factors [4,12]. Like other marine organisms, yellow clams can be susceptible to several factors that contribute to their mortality, which can be categorized into environmental, biological, and anthropogenic causes [3,13,14,15,16,17,18,19]. While the exact causes of the mass mortality events affecting A. mactroides remain unknown [4,11,12], this species has been considered endangered since the 1990s [11,20]. There is therefore an urgent need to ensure the conservation of natural populations of this species, which requires a multifaceted strategy that combines ecological protection, sustainable resource management, pollution control and community engagement [6,9,10,21,22,23]. The development of technology to produce juvenile clams in captivity could be complementary to these approaches, as it would allow for evaluating the viability of both restocking natural environments and producing clams in sustainable aquaculture systems.

A. mactroides is a dioecious species, with no apparent sexual dimorphism. It reproduces sexually through the release of large quantities of gametes into the water column, where fertilization and the formation of planktonic larvae occur [3]. At the end of larval development, yellow clams bury themselves in sandy bottoms [3,24]. Sexual maturation in A. mactroides begins in the first year of life, with the peak of spawning normally occurring during the winter–spring periods [24,25]. Under laboratory conditions, reproduction of this species has only been possible through gamete stripping [3,26], a procedure that in addition to sacrificing the breeders can negatively affect the fertilization, survival, growth, and the duration of the larval period [27,28]. It is therefore important to overcome this bottleneck as there are records of successful spawning of other members of the Mesodesmatidae family under hatchery settings [29,30].

In the process of producing bivalve seeds in captivity, the conditioning of broodstock is a fundamental step that consists of maintaining the animals under optimal environmental conditions for an adequate period of time to ensure that a larger proportion of the broodstock will be fully mature and thus available for reproductive purposes [31,32,33,34,35]. Conditioning usually maximizes the fecundity of the broodstock while maintaining the quality of the oocytes and, ultimately, larval viability. An array of synergistic, intrinsic and extrinsic factors play an important role in achieving fully mature individuals. The specific endogenous rhythms that regulate the reproductive cycle are synchronized by external factors, particularly temperature, animal health and nutritional status [36,37]. Here, we evaluated the duration of the conditioning period that would result in the highest proportion of mature yellow clam breeders, which may facilitate assisted reproduction of this species in captivity.

2. Materials and Methods

2.1. Origin and Management of Broodstock

An initial sample of 250 yellow clams visually measuring more than 50 mm in length was collected on 7 July 2023, in the intertidal zone of Cassino beach, Rio Grande, southern Brazil. At the time of collection, water temperature and salinity were measured in situ with a thermometer and optical refractometer, respectively. The geographic coordinates of the collection station (32°12′30.1″ S, 52°10′33.9″ W) were taken with a handheld navigator (GPS; model Garmin eTrex). The clams were taken from a single population to minimize the influences of genetic variability and location on reproductive condition. They were immediately transported to the laboratory in plastic containers containing sand and seawater. Brazilian legislation requires no authorization from ethics or animal welfare committees for research on invertebrates [38].

In the laboratory, a subsample of 30 clams was randomly selected, dried on paper towels, and their total length (L), total shell height (H) and width (W) were measured with a vernier caliper (0.01 mm accuracy; Vonder, Brazil). The wet weight of the whole clam (WWc), soft tissues (WWst), and valves (WWv) was obtained with an electronic scale (0.01 g precision; Marte Científica, Brazil). The dry weight of soft tissues (DWst) and valves (DWv) was determined after 48 h in an oven at 60 °C and 24 h in a desiccator. Additionally, the condition index (CI) was estimated according to the following formula [39]:

CI = (DWst/(WWc − WWv)) × 100

(1)

Clams with broken valves, siphons or feet without movement, and with L smaller than 50 mm, were discarded. A total of 160 clams were then transferred to the maintenance system.

2.2. Maintenance System and Experimental Design

The experimental set-up used for clam maintenance, feeding and management followed that used by Gauthier et al. [40,41]. Eight clams were stocked per experimental unit (EU), which consisted of a 15 cm diameter weldable PVC pipe and coupling, containing a 10 cm layer of sterilized beach sand over a 200 µm mesh. EUs were placed in 500 L tanks filled with filtered (1 µm) and chlorinated/dechlorinated seawater. The temperature in the tanks was maintained between 15 °C and 20 °C using chillers or immersion heaters as required. Each EU was equipped with an air–water pump (airlift) that forced seawater through the sand layer from top to bottom. Around 20% of the water of all tanks was renewed daily. Water quality was monitored by daily measurements of dissolved oxygen concentration, temperature and salinity with a multiparameter probe (YSI 550 A, Yellow Springs Instruments, Yellow Springs, OH, USA) and a handheld refractometer (ATC, Kasvi, Pinhais, Brazil), respectively. Values of pH and concentrations of total ammonia [42] and nitrite [43] were measured twice a week, while nitrate concentrations were measured once a week [44].

Based on the results of previous studies [40,41], clams were fed a commercial concentrate of the microalgae Isochrysis galbana and Chaetoceros muelleri at a ratio of 70:30 (MixFresh, AlgaSul, Rio Grande, Brazil). A concentration of 14 × 10⁴ cells·mL⁻¹ was maintained through daily monitoring using a Neubauer chamber. When the concentration fell below the pre-established level, more microalgae were added. Microalgae consumption per clam (C; cells·mL⁻¹·clam⁻¹) was estimated using the following formula:

C = (14 × 10⁴ − MC)/n

(2)

where 14 × 10⁴ is the established initial concentration of microalgae, MC is the total number of microalgae cells at the morning count, and n is the number of live clams per EU.

The clams were kept in the laboratory for different conditioning periods: 14, 28, 45 or 60 days. Twenty EUs were used, with four replicates for freshly collected clams (day 0) and for each of the four conditioning periods. On days 0, 14, 28, 45, and 60, the clams were subjected to a spawning induction process by means of thermal shocks and the addition of sperm to the water, which is considered the most promising stimulus to induce spawning in A. mactroides [41]. For this purpose, the EUs were transferred to 18 L containers maintained at 26 ± 1 °C, where they remained for 24 h. The addition of sperm followed Argüello-Guevara et al.’s procedure [45]. Each day that spawning induction was performed, sperm obtained from 2 to 4 wild clams collected at the previously georeferenced collection station was pooled and transferred to a 1 L beaker at a density of 10 million sperm.mL⁻¹ for the purpose of providing pheromones [41,46]. Sperm motility was assessed after activation with seawater under a light microscope (200×). Approximately 100 mL of the sperm solution was added to the 18 L containers every 30 min for 2 h. The water temperature was kept constant with immersion heaters. Nearly 24 h after the spawning induction process, all eggs and trocophore larvae present in 20 mL samples were counted under an optical microscope using 0.2 mL volume slides. This procedure was carried out for two consecutive days.

Groups of 8 clams, 2 from each of the four replicates, were sampled on the day of collection (day 0) and after 14, 28, 45, and 60 days of conditioning in the laboratory, but before spawning induction. Their tissues were fixed in 20% saline formaldehyde for histological analysis. On the same days (days 0, 14, 28, 45 and 60), 12 wild clams were collected in the previously georeferenced collection station and their tissues were also fixed for histology.

2.3. Histological Analysis

Clam tissues fixed in 20% saline formaldehyde were cut, placed in labeled cassettes and processed in a Leica TP1020 automatic processor (Leica, Wetzlar, Germany). The blocks were embedded in paraplast and sectioned at 5 µm with a Leica RM 2245 microtome (Leica, Wetzlar, Germany). Hematoxylin and eosin-stained slides were examined in a Zeiss Primo Star optical microscope equipped with a camera and AxioVision 4.8.2 software (Carl Zeiss, Oberkochen, Germany) and processed using Adobe Photoshop CC 2024 (Adobe Inc., San Jose, CA, USA) for linear adjustments of contrast and brightness. The developmental stages of the gonads were classified according to Herrmann et al. [24]: rest—RE, early gametogenesis—EG, advanced gametogenesis—AG, mature—MT, spawning—SP, spent—ST, and recovery—RC.

The largest diameter (D) of 20 to 50 oocytes with visible nuclei from each female were measured using the ImageJ1 software version 1.53t (National Institutes of Health, Bethesda, MD, USA). Based on previous results [41,47], the proportion (%) of oocytes with diameters greater than 45 µm was estimated as a proxy of female clams ready to spawn.

2.4. Data Analysis

Each EU was considered an independent replicate. Since the yellow clam does not exhibit apparent sexual dimorphism and assuming a 1:1 sex ratio, eight clams were placed in each EU. Thus, each EU would theoretically present a balanced proportion of males and females, allowing spawning to occur in each replicate. Egg and/or trochophore larvae counts were quantified and analyzed at the EU level, as spawning, fertilization, and initial larval development occurred in each EU. Measurements of the oocyte diameter were taken from a single clam of the two individuals sampled from each replicated EU on days 0, 14, 28, 45 and 60.

The number of eggs/trocophore larvae and the diameter of oocytes from wild and laboratory-conditioned clams were tested for normality and homogeneity of variances using the Shapiro–Wilk and Levene tests, respectively [48]. As the assumptions for the number of eggs/trocophore larvae were not confirmed, the data were subjected to nonparametric analysis and the Kruskal–Wallis test to verify the effect of conditioning time on gonad development. On the other hand, once the premises for the oocyte diameter were confirmed, data were subjected to analysis of variance (ANOVA) to verify the effect of the duration of the conditioning period. If there were differences, Tukey’s a posteriori test for multiple comparison was performed. Percentage data were normalized by square root-arcsine, but only non-transformed means are presented. All analyses were performed with a 95% significance level. Results are presented as mean ± standard deviation (±SD).

3. Results

The mean L of clams collected on day 0 (7 July 2023) was 63.94 (±4.91) mm; H = 33.41 (±2.58) mm; W = 15.35 (±1.13) mm; WWc = 8.60 (±2.25) g; WWst = 4.14 (±1.09) g; WWv = 2.61 (±0.89) g; DWst = 0.78 (±0.19) g; and DWv = 2.06 (±0.77) g. The condition factor was estimated at 13.46 (±2.63).

The water temperature at the time the clams were collected in the wild was 16.0 °C (days 0 and 14), 16.5 °C (day 28), 14.0 °C (day 45) and 15.5 °C on day 60. Temperature in the EUs varied from 15 to 20 °C with a mean of 16.7 °C (±1.8). On day 21, a drop to 10.0 °C occurred for a period of 24 h due to a power failure, but no major impacts are expected, as all EUs were affected equally. Also, 10 °C is within the temperature range that occurs in the geographical distribution area of the yellow clam. Dissolved oxygen concentrations remained above 5.77 mg.L⁻¹ throughout the experimental period (mean of 7.32 ± 0.91 mg.L⁻¹). The concentration of total ammonia varied from 0.07 mg.L⁻¹ to 0.41 mg.L⁻¹; nitrite from 0.01 to 0.04 mg.L⁻¹; and nitrate between 0.00 and 0.08 mg.L⁻¹. The pH remained between 7.93 and 8.34. The mean survival rate of clams maintained in the laboratory for 14, 28, 45 and 60 days was estimated at 100%, 100%, 99.4% and 96.9%, respectively.

The mean daily consumption of the microalgae concentrate (mix of I. galbana and C. muelleri) throughout the experimental period was estimated at 395 (±73) cells.mL⁻¹.clam⁻¹ (Figure 1).

On day 20, when there was a power outage and the water temperature dropped to 10.0 °C, microalgae consumption decreased to the lowest value of 132 cells.mL⁻¹.clam⁻¹. The highest consumption was 568 cells.mL⁻¹.clam⁻¹ on day 53 (Figure 1).

Histological examination of clam tissues confirmed the occurrence of distinct stages of gonadal development in both sexes: rest (RE), early gametogenesis (EG), advanced gametogenesis (AG), mature (MT), spawning (SP), spent (ST) and recovery (RC). In the early stage of gametogenesis, males had gonads with small follicles and a low presence of germ cells (Figure 2A), while females had oogonia and primary oocytes (Figure 3A). As gametogenesis progressed, males presented follicles with increased volume and a few sperm cells (Figure 2B). In females, some mature oocytes with a polygonal shape were observed (Figure 3B). Mature males (Figure 2C) had follicles full of sperm, while mature females presented free polygonal oocytes within the follicles (Figure 3C). Males and females in the SP stage (Figure 2D and Figure 3D, respectively) presented empty spaces between mature gametes and ruptured reserve tissue with a flaccid appearance. Regardless of sex, the follicles of spent clams were small, practically empty and with few degraded residual gametes (Figure 2E and Figure 3E). Both male and female clams in the RC stage presented a high presence of connective tissue and few follicles with a smaller diameter (Figure 2F and Figure 3F). Clams in the RE stage were characterized by the total absence of gametes, which made it impossible to distinguish sex (Figure 4).

Figure 5 highlights histological sections of mature yellow clam gonads after 45 days of conditioning in the laboratory. In the male, testicular tissue with abundant spermatocytes is clearly observed, while the ovary presents several mature polygonal oocytes, interstitial cells, and some remaining immature follicles.

The proportion of gonadal stages of female and male clams collected in the wild and conditioned in the laboratory is presented in Figure 6. The gonads of female clams collected on day 0 were in the RE (25%) or EG (75%) stages, while males were also in the RE (20%) or EG (80%) stage. Regardless of sex, there was an increase in the proportion of clams with more advanced stages of gonadal development as the experimental period progressed, both in wild and laboratory-conditioned individuals.

Figure 6 shows the proportional increase in gonads in the MT and SP stages of development in comparison to the total number of individuals sampled at each time point (0, 14, 28, 45 and 60 days) over the experimental period. No female or male clams had gonads in the MT or SP stages of development on days 0 and 14. On day 45, however, all males and females conditioned in the laboratory had gonads in the MT stage (Figure 5, Figure 6 and Figure 7). At the end of the experiment (day 60), all wild males were mature, while those conditioned at the laboratory were mature (75%) or in recovery (25%). Wild females on day 60 presented gonads in the stages of AG (25%), MT (50%) or SP (25%). Gonads of laboratory-conditioned females were either in the MT (50%) or RC (50%) stages of development.

The proportion of the different stages of gonadal development of female and male clams in the experimental units where spawning occurred after induction is shown in Figure 8. Most gonads were in relatively advanced stages of development (MT and SP) or had recently spawned (ST or RC). Irrespective of the number of days of conditioning (28, 45 or 60 days), at least 50% of the females had gonads in the spawning stage of development.

No spawning was observed in clams collected in the wild and induced to spawn on day 0, as well as in those conditioned in the laboratory for 14 days (

Table 1. Number of eggs/trocophore larvae (×10³) per experimental unit (EU) containing eight adults of the yellow clam (Amarilladesma mactroides) on the day of collection (day 0) at Cassino beach, southern Brazil, and after 14, 28, 45 and 60 days of conditioning in the laboratory followed by spawning induction with thermal shock and exposure to sperm solution.

	Day
	0	14	28	45	60
EU 1	0	0	0	0	0
EU 2	0	0	90.0	832.0	180.0
EU 3	0	0	90.0	0	90.0
EU 4	0	0	720.0	270.0	130.0
Mean (±SD)	0 (±0)	0 (±0)	225 (±333)	276 (±392)	100 (±76)
Total	0	0	900.0	1102.0	400.0

). The occurrence of spawns, however, was observed after 28, 45 and 60 days of conditioning in the laboratory in at least two of the four experimental units. Although the total number of eggs/trocophore larvae estimated for the group of clams conditioned for 45 days (1102 × 10³) was numerically higher than in the other treatments, no significant differences (Χ² = 5.00, df = 3; p = 0.17) were detected between conditioning periods (Table 1). This was likely due to the high variation observed within means, particularly 28 days and 45 days of conditioning. For these treatments, the coefficient of variation, defined as the ratio of the standard deviation to the mean, was estimated at 148% and 142%, respectively. The occurrence of trocophore larvae was observed in most treatments after a few hours.

The mean oocyte diameter (D) and the proportion (%) of oocytes with diameters greater than 45 µm (D ≥ 45 µm) from wild clams collected on days 0, 14, 28, 45 and 60 and those of the wild clams that were conditioned in the laboratory for 14, 28, 45 and 60 days are presented in

Table 2. Mean (±SD) oocyte diameter (D; µm) and proportion (%) of oocytes greater than 45 µm (D ≥ 45 µm) of wild yellow clam (Amarilladesma mactroides) collected at Cassino beach, southern Brazil, on days 0, 14, 28, 45 and 60, and of wild clams that were conditioned in the laboratory for 14, 28, 45 and 60 days. Superscript capital and lower-case letters indicate significant differences within rows and within columns, respectively (p < 0.05).

	Wild (n = 5–8)	Laboratory-Conditioned (n = 3–4)
D
Day 0	19.88 ± 4.08 d
Day 14	22.68 ± 4.07 A,c	22.76 ± 4.08 A,d
Day 28	34.37 ± 3.95 B,b	36.33 ± 4.07 A,c
Day 45	44.74 ± 4.05 A,a	45.14 ± 4.04 A,a
Day 60	45.85 ± 4.09 A,a	43.45 ± 4.07 B,b
D ≥ 45 µm
Day 0	0.0 ± 0.0 y
Day 14	0.0 ± 0.0 X,y	0.0 ± 0.0 X,z
Day 28	0.0 ± 0.0 X,y	3.13 ± 5.43 X,z
Day 45	45.00 ± 5.77 Y,x	53.47 ± 4.50 X,x
Day 60	55.20 ± 12.71 X,x	33.72 ± 4.31 Y,y

. The diameter of oocytes increased significantly over time for both clams collected in the natural environment and those conditioned in the laboratory. Clams conditioned in the laboratory for 28 days had significantly greater oocytes (mean diameter = 36.33 µm) than those collected in the wild on the same day (34.37 µm), but the opposite was observed on day 60, when wild-collected clams had significantly greater mean oocyte diameters (45.85 µm vs. 43.45 µm). A similar trend was observed for the proportion of oocytes with D ≥ 45 µm. Among laboratory-conditioned clams, oocytes with D ≥ 45 µm were observed from day 28 onwards, while wild-collected clams only presented oocytes greater than 45 µm on days 45 and 60. The proportion of oocytes with D ≥ 45 µm in laboratory-conditioned clams was significantly higher on day 45. In wild clams, no significant difference was observed in the proportion of oocytes with D ≥ 45 µm between days 45 and 60.

4. Discussion

Histological examination demonstrated that the gonadal tissues of A. mactroides of both sexes could be divided into seven distinct stages of development: rest, early gametogenesis, advanced gametogenesis, mature, spawning, spent and recovery, which somewhat agrees with previous studies [24,25,40,41,49,50,51]. Histological analysis revealed that the gonads of wild-collected clams on day 0 were in the resting or early gametogenesis stage. Although oocyte size increased significantly over time for laboratory-conditioned clams as well as for those in the wild, the oocyte diameter of wild-collected clams on day 0 (mean 19.88 µm) was much smaller than that considered necessary for mature, spawn-ready yellow clams. The minimum diameter of mature oocytes is within the range of 45 µm to 55 µm [24,26,41,47,49]. Therefore, it is not surprising that the clams collected on day 0 failed to liberate gametes when induced to spawn. Based on this, it can be concluded that, at the beginning of the experimental period, the gonads of wild-collected clams were homogeneously distributed in the early stages of development.

The first spawns were only observed on the 28th day of conditioning, when 900 × 10³ eggs/trocophore larvae were obtained. Histology of the gonads demonstrated that a significant portion of these clams were mature (50% of males and 33% of females) and therefore capable of spawning. Measurement of the oocytes indicated that, at 28 days, their mean diameter (36.33 µm) was below what is considered the minimum for a female to spawn (≥45 µm). However, because a small proportion of these oocytes (3.13%) had a diameter greater than 45 µm, some females were already capable of releasing gametes. These results, therefore, indicate that, in the case of A. mactroides and under the present experimental conditions, adult clams may require a minimum of 28 days of laboratory conditioning to reach gonadal maturity and spawn. However, after 45 days of conditioning, it was histologically demonstrated that all female and male clams had mature gonads, and furthermore, the mean oocyte diameter (45.14 µm) was larger than the minimum size for a female to be considered ready to spawn. Not surprisingly, by day 45, a total of 1102 × 10³ eggs were released after spawning induction. All of this suggests that a conditioning period longer than 28 days may result in a higher proportion of the broodstock with fully mature gonads and therefore available to produce offspring. This is in agreement with an early study [33] which recommended that conditioning of temperate and cold-water climate bivalves should take 4 to 8 weeks. The present results also agree with previous ones [41], when an increase in oocyte size and more advanced stages of gonadal development in wild-collected A. mactroides were reported after 14 days of laboratory conditioning. These authors concluded that the 14-day conditioning period was not sufficient for the complete development of the gonads. In a study with Mesodesma donacium, a clam species from the same family as A. mactroides, the highest proportion of mature clams (56%) was observed after 44 days of conditioning [52]. It should be considered, however, that the duration of the conditioning period will depend on the maturation stage of the individuals at the time of collection [33], whether in a natural environment or in cultivation structures. Environmental conditions such as temperature, photoperiod and salinity, and the quantity and quality of the food, are commonly identified as regulatory factors for gonadal maturation in bivalves [32,33,34,37,53,54], with temperature and food availability being the key factors that affect the reproductive cycle [28,55]. Temperature is recognized as the main parameter affecting the reproductive development of bivalves, having important implications for the success of broodstock conditioning in a hatchery environment [34,56]. Food availability influences energy storage and use, affecting gonadal development, oocyte quality, and larval viability [32,57].

Maintaining the broodstock in the laboratory for a longer period, in this case 60 days, produced no positive effects. Histological analysis indicated that, on day 60, half of the females were in the recovery stage, and therefore a significant decrease in mean oocyte diameter was observed. Although a total of 400 × 10³ eggs were spawned, our results suggest that under the conditions of this study, 60 days may be too long, as some individuals likely spawned earlier or reabsorbed gametes that were not released.

Since maintaining broodstock in bivalve hatcheries is an expensive process, mainly due to the cost of microalgae production [58,59,60,61], an alternative strategy that could be considered is the periodic monitoring of gonadal development of clams, regardless of whether they are in the natural environment or in aquaculture settings. When the clams are observed to have mature gonads or are close to reaching an advanced stage of gonadal development, collection and acclimation to laboratory conditions could then be performed. Knowing when clams are approaching or entering peak gonadal maturity would not only minimize the need for conditioning in captivity but would also help synchronize hatchery operations through the alignment of broodstock collection operations with peak gonadal maturity. Avoiding the use of clams with regressed or immature gonads, as was the case here with clams kept in the laboratory for 60 days, would positively affect reproductive output in terms of larval quality and quantity, as individuals at peak maturity can be selected for spawning, ensuring high fertilization rates and possibly healthier larvae.

On day 28, the higher proportion of laboratory-conditioned clams in more advanced stages of gonadal development (mature or spawning) compared to wild individuals and the occurrence of spawning in clams conditioned in the laboratory suggest that maintenance in captivity accelerated gonadal development compared to the wild. This hypothesis is supported by the significantly larger mean oocyte diameter in laboratory-conditioned clams and the initial occurrence of oocytes with diameters greater than 45 µm, which is indicative of females ready to spawn. Earlier spawning for clams is possible, but it essentially depends on environmental factors and the consistent provision of high-quality diets [33,62,63,64]. Under controlled laboratory or hatchery conditions, adjusting certain environmental cues, such as water temperature, salinity, and photoperiod, and maintaining these environmental parameters stable over time, as well as providing adequate amounts of nutrient-rich diets, can stimulate earlier gonadal development and accelerate the reproductive process, causing spawning to occur earlier than would otherwise occur naturally [63]. A strategic advantage of achieving earlier spawning is the possibility of having clams in spawning condition for most of the year, thus extending the period in which hatcheries have access to larvae [33].

After clams were induced to spawn through thermal shock and exposure to the sperm solution, a significant portion still had gonads in relatively advanced stages of development (mature and spawning) or had recently spawned (spent or recovery). Irrespective of the duration of the conditioning period (28, 45 or 60 days), we found that at least 50% of the females that were exposed to spawning induction had gonads in the spawning stage of development, clearly indicating the possibility of new spawns occurring in the coming days. This points to the need to maintain continuous monitoring of the occurrence of spawning even 48 h after exposure to the induction procedures, as initially defined in this study. The possibility of spawning occurring even after 48 h of monitoring indicates that the total spawning values reported here may have been underestimated. This is a topic of ongoing research in our group.

Future studies should also consider the possible effects of broodstock conditioning on offspring quality. Crisóstomo et al. [65] reported that both wild- and laboratory-conditioned scallops (Argopecten purpuratus) produced viable gametes and larvae, but oocytes and larvae from wild-conditioned scallops were larger and had higher lipid content. The fact that these parameters were lower in laboratory-conditioned individuals highlights the importance of further optimizing broodstock conditioning protocols in captivity, which would allow for more predictable and sustainable production.

5. Conclusions

Our results indicate that conditioning for 45 days resulted in 100% of the yellow clams reaching gonadal maturity. When exposed to a non-invasive method of spawning induction (combined exposure to thermal shock and sperm), several clams responded by releasing gametes, allowing larvae to be obtained. Furthermore, the high survival rate of clams kept in captivity for 60 days (>95%) and the occurrence of multiple spawns reinforce the notion that the experimental system, environmental conditions, and feeding management were able to minimally replicate key aspects of the natural habitat conducive to the reproduction of A. mactroides in captivity. These results provide a better understanding of the environmental requirements and management practices that will allow for the controlled reproduction of this important bivalve species.