Gonad Status, Condition Index and Biochemical Composition of the Yellow Clam Amarilladesma mactroides

Abstract

The yellow clam, Amarilladesma mactroides, is an abundant component of the intertidal infauna of the southwestern Atlantic Ocean and an important source of food and income for local communities. In recent years, this species has been increasingly affected by anthropogenic and environmental pressures, contributing to the decline and collapse of several populations. In this study, we characterized the reproductive cycle of A. mactroides in the southernmost region of Brazil using standard approaches, including gonadal histology and biometric parameters, complemented by analyses of proximal composition and fatty acid profiles. Monthly samples of ≥100 clams were collected from October 2021 to September 2022. The results revealed two distinct reproductive peaks: a major peak from October to December (austral spring–summer) and a less pronounced peak from May to July (autumn–winter). Although gonadal histology, particularly oocyte diameter, provided the primary evidence for defining these reproductive peaks, the condition index and concentrations of total lipids and n-3 polyunsaturated fatty acids (n-3 PUFA) also proved useful indicators of reproductive activity. These findings provide new insights into the reproductive dynamics of A. mactroides in southern Brazil and may contribute to the development of strategies for the sustainable management, conservation and aquaculture of this ecologically and socioeconomically important bivalve.

1. Introduction

The yellow clam, Amarilladesma mactroides (Reeve 1854), occurs naturally in the infralittoral zone of sandy beaches from northern Argentina to Rio de Janeiro, Brazil, where it is often the dominant mollusk species in terms of biomass [1,2]. Its exploitation as a food resource dates back at least 4000 years among the coastal populations of southern Brazil [3] and Argentina [4]. More recently, A. mactroides has also been used as bait in recreational fisheries [5]. However, intensive harvesting has contributed to the collapse of several populations throughout its distribution range [1,2,5]. In addition, A. mactroides is highly susceptible to pollution and other anthropogenic disturbances [6,7,8,9] and is strongly affected by climate change [10]. Together, these pressures have caused substantial changes in population abundance and structure, raising concerns about the long-term conservation and sustainable use of this ecologically and socioeconomically important bivalve.

In natural environments, the reproductive cycle of A. mactroides is primarily regulated by temperature [1,10,11]. Along the Argentine coast, which represent the southern limit of the species’ distribution, spawning has been reported from late November to mid-March [1], whereas two distinct reproductive periods have been identified, with a major event in spring and a less intense event in summer [12]. In contrast, two annual reproductive events occurring in winter and spring have also been described [13]. These contrasting findings suggest considerable spatial variability in reproductive dynamics across the species’ range, likely driven by regional differences in environmental conditions, particularly temperature regimes and food availability [14].

Although the reproductive cycle of a population from Santa Catarina, Brazil, has recently been described [15], reproductive patterns in bivalves are strongly influenced by local environmental conditions [14]. Therefore, information from a single population is insufficient to characterize the reproductive dynamics of the species across its entire distribution range. This limitation is particularly relevant for southern Brazil, where Cassino beach constitutes one of the major remaining strongholds of A. mactroides. Extending for more than 220 km, Cassino beach encompasses an extensive dissipative sandy-beach ecosystem, which corresponds to the preferred habitat of the species. Consequently, populations occurring in this region may play an important role in maintaining population connectivity and species persistence throughout its geographic range [16]. Despite its ecological significance, however, the reproductive biology of this population remains poorly understood. Further studies are therefore needed to evaluate geographic variation in reproductive traits and to provide a stronger scientific basis for the management, conservation, and potential aquaculture of this species [17]. In this context, an important question is how the reproductive cycle of A. mactroides in southern Brazil varies throughout the year and how gonadal development is reflected in its biometric condition and biochemical composition.

The condition index (CI) is widely used as an indirect indicator of reproductive status in bivalves because it reflects changes in soft-tissue biomass associated with gonadal development [18]. In general, CI values increase as gametogenesis progresses and gonadal mass expands during gamete maturation. This process is frequently accompanied by the accumulation of biochemical reserves, particularly lipids and proteins, within the developing gonads [18,19]. Consequently, CI values typically reach their maximum during gonadal maturity and decline following spawning as gametes are released. However, because CI provides only an indirect measure of reproductive condition, histological analysis remains essential for accurately characterizing gonadal development and spawning activity. The combined assessment of gonadal stage, CI, and biochemical composition can therefore provide a more comprehensive characterization of the reproductive cycle and the physiological changes associated with gametogenesis and spawning.

The present study aimed to improve our understanding of the reproductive biology of A. mactroides in southern Brazil by combining histological assessment of gonadal development with biometric, condition index, and biochemical analyses throughout an annual cycle. Specifically, we characterized seasonal changes in gonadal development and reproductive activity and evaluated their relationships with the condition index and biochemical composition. By identifying periods of gametogenic development, maturation, and spawning, this work provides information that is fundamental for the sustainable management and conservation of natural populations. Furthermore, the findings may contribute to the development of aquaculture technologies for the species, as successful hatchery production depends on a thorough understanding of its reproductive cycle and associated physiological changes.

We hypothesized that the reproductive cycle of A. mactroides in southern Brazil is characterized by distinct seasonal patterns of gonadal development and spawning, and that these changes are reflected in the condition index and biochemical composition. Specifically, we predicted that the condition index and the concentrations of biochemical reserves, particularly protein and lipids, would increase during gametogenesis and gonadal maturation, reach higher values around reproductive maturity, and subsequently decrease following spawning as endogenous reserves are mobilized and gametes are released. We further hypothesized that the magnitude and timing of these changes would be associated with seasonal variations in environmental conditions, particularly temperature and food availability.

2. Materials and Methods

2.1. Sourcing and Measurements

In the last week of each month from October 2021 to September 2022, at least 100 yellow clams were collected in the infralittoral zone of Cassino beach, municipality of Rio Grande, southern Brazil, using flat shovels. Water temperature, salinity, pH, and dissolved oxygen concentrations were measured in situ with a thermometer, an optical refractometer (Atago, Tokyo, Japan), and a portable multiparameter device (YSI 550 A, Yellow Springs Instruments, Yellow Springs, OH, USA), respectively. Clams were immediately transported to the laboratory in thermal boxes containing seawater collected at the sampling site. The transportation time was less than 30 min.

Upon arrival at the laboratory, clams with broken valves or with immobile siphons or feet were discarded. Only adult clams (≥50 mm) were selected [1]. The clams were sacrificed by immersion in an ice/water bath (1:1) and blotted dry, and the total length (L), total height (H), and width (W) of the shell were measured to the nearest 0.01 mm with a manual caliper (Vonder, Curitiba, PR, Brazil). The wet weight of the whole clam (WWc), soft tissues (WWst), and valves (WWv) were obtained using an electronic scale (0.01 g precision; Marte Científica, São Paulo, SP, Brazil). The dry weights of the soft tissues (DWst) and valves (DWv) of 20 individuals per month were measured after 48 h in an oven at 60 °C, followed by 24 h in a desiccator. The condition index (CI) [CI = (DWst/DWv) × 100] was estimated [18].

2.2. Immunological Analyses

Hemolymph was sampled from the pericardial cavity of 10 clams collected each month using a 1 mL syringe filled with heparin (Hepamax-S, Blau Farmacêutica, Cotia, SP, Brazil) as an anticoagulant. For differential hemocyte count (DHC), an aliquot of hemolymph was smeared onto a slide fixed in methanol for 10 min. The slides were then stained with May Grunwald Giemsa absolute for 2 min, washed with distilled water for 2 min, soaked in 20% May Grunwald Giemsa for 10 min, and washed again with distilled water [20]. Another aliquot of the hemolymph samples was mixed in a 1:1 ratio with a PBS solution (NaCl 0.137 M, KCl 2.7 mM, KH₂PO₄ 1.5 mM, Na₂HPO₄ 8.1 mM, CaCl₂ 0.9 mM, MgCl₂ 0.49 mM, pH 7.4), plus 0.178 g Congo Red and 1.5 g biological yeast to estimate the number of phagocytic hemocytes. The numbers of hyaline (HH) and granular (GH) hemocytes, as well as phagocytic hemocytes, were determined using an optical microscope at 40× magnification (Nikon Eclipse e200, Sendai, Japan). The results are expressed as percentages (%).

The total concentration of proteins in the hemolymph (HPC) was determined using a commercial kit (Bioclin K031, Química Básica Ltd., Belo Horizonte, MG, Brazil). Absorbance was measured at 545 nm using a microplate reader (BioTek Synergy HT, Winooski, VT, USA).

2.3. Gonad Histology

Histological analyses were conducted exclusively on female clams to provide a more accurate assessment of the gametogenic development. Gonadal stages were characterized according to previously established criteria [13]. Approximately 1 cm³ of gonadal tissue was excised with a scalpel, placed in histological cassettes, dehydrated through a graded ethanol series, embedded in Paraplast paraffin wax (Sigma-Aldrich, Saint Louis, MO, USA), sectioned at 3–5 μm using a Leica RM2245 microtome (Leica, Wetzlar, Germany), and stained with hematoxylin and eosin following standard histological techniques. Oocyte diameters were measured only for oocytes with a visible nucleus, corresponding to sections passing through the approximate center of the oocyte. Measurements were performed for each female using a Nikon Eclipse e200 microscope (Nikon, Sendai, Japan) equipped with an eyepiece reticle calibrated against a stage micrometer.

2.4. Biochemical Composition

Prior to sampling the soft tissues, intestinal contents were evacuated to avoid affecting the biochemical composition results. Seawater was gently injected into the digestive tract of the clams using 0.05 mL syringes. Crude protein concentration in the soft tissues was determined by the Dumas combustion method (AOAC method 990.03) [21] using a Leco nitrogen/protein analyzer (Leco Corporation, St. Joseph, MO, USA). Total lipids were quantified as ether extract by Soxhlet extraction according to AOAC method 920.39C [21]. Fatty acid composition was determined by gas chromatography. Fatty acid methyl esters were prepared following a standard transesterification procedure [22] and separated using a gas chromatograph (Agilent 7890B, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and a CP-Sil 88 capillary column for FAMEs (60 m × 0.25 mm × 0.20 µm; CP7487, 7-inch cage). The chromatographic conditions were as follows: detector temperature of 300 °C; injector temperature of 240 °C; initial column temperature of 120 °C for 10 min, programmed to increase at a rate of 3 °C min⁻¹ up to 186 °C, held for 5 min, and then increased again at 3 °C min⁻¹ up to the final temperature of 231 °C. Helium was used as the carrier gas, with a column flow rate of 2.5 mL min⁻¹, and the injection was performed in split mode (1:40). Fatty acids were identified based on the retention time of two standards: MIX 37 (37 Component FAME Mix and PUFA n⁰ 3) and menhaden oil (Supelco, Bellefont, PA, USA). The fatty acid concentrations in the samples were then calculated using 23:0 tricosanoic acid (Sigma, Saint Louis, MO, USA) as an internal standard [23]. The peak areas were corrected using the theoretical relative FID response factor [24].

Lactate levels in the hemolymph were determined in four replicates per month using a commercial kit (Bioclin K082, Química Básica Ltd., Belo Horizonte, MG, Brazil). The absorbance was recorded at 340 nm using a microplate reader (BioTek Synergy HT, Winooski, VT, USA). To determine the glycogen content in the hemolymph, four replicate samples per month were boiled for 10 min in KOH 30% with a saturated 5% Na₂SO₄ solution [25]. After cooling, 95% ethanol was added, and the samples were allowed to rest for 40 min. The resulting precipitate was resuspended in distilled water, followed by the addition of 95% ethanol. After 90 min, the precipitate was collected and used for glucose measurement using a commercial kit (Bioclin K084, Química Básica Ltd., Belo Horizonte, MG, Brazil). Absorbance was measured at 505 nm using a microplate reader (Biotek Synergy HT, Winooski, VT, USA).

2.5. Statistical Analyses

The homogeneity of variances and normality of data distribution were assessed using Levene’s and Shapiro–Wilk tests, respectively. One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used to evaluate differences in condition index (CI), crude protein, and total lipid concentrations among sampling months. A nested ANOVA was performed with oocyte measurements nested within females to assess differences in oocyte diameter among sampling months. The nonparametric Kruskal–Wallis test was used to evaluate temporal variations in biometric parameters (L, H, W, WWc, WWst, WWv, DWst, and DWv), glycogen and lactate concentrations, fatty acid profiles, and hemocyte counts when the assumption of normality and homogeneity of variances were not met. When significant differences were detected by the Kruskal–Wallis test, Dunn’s multiple comparisons test was applied. All statistical analyses were conducted at a significance level of 5%.

3. Results

The minimum and maximum temperatures during the sampling period (9.8 °C and 28.2 °C) were observed in June and February 2022, respectively, whereas the highest salinity (38) occurred in December 2021 and January 2022, and the lowest (28) in June 2022 (

Table 1. Temperature (°C), salinity, dissolved oxygen concentration (DO; mg L⁻¹) and pH of seawater at the time of sampling of yellow clam Amarilladesma mactroides on Cassino beach, southern Brazil, from October 2021 to September 2022 (nd = not determined).

	Temperature	Salinity	DO	pH
October 2021	21.5	34	8.46	8.06
November 2021	21.7	35	7.80	8.32
December 2021	23.7	38	7.34	7.80
January 2022	26.0	38	6.32	7.84
February 2022	28.2	37	6.34	7.84
March 2022	19.5	37	6.64	8.69
April 2022	20.2	33	6.31	8.28
May 2022	16.3	32	8.12	7.65
June 2022	9.8	28	8.76	nd
July 2022	15.0	35	7.54	8.32
August 2022	15.3	32	9.04	7.89
September 2022	15.0	32	8.80	7.65

). The DO concentration remained above 6.3 mg L⁻¹ throughout the sampling period. The pH varied from 7.65 in May and September 2022 to 8.69 in March 2022.

Although the biometric parameters (L, H, W, WWc, WWst, DWst, WWv, and DWv) varied throughout the sampling period (

Table 2. Mean (±SD) length (L), height (H), width (W), total wet weight (WWc), wet weight and dry weight of the soft tissues (WWst and DWst, respectively), and wet and dry weights of the valves (DWv and DWv, respectively) of the yellow clam Amarilladesma mactroides sampled at Cassino beach, southern Brazil, from October 2021 to September 2022. Values in parentheses represent sample numbers (n). Within columns, superscript letters indicate significant differences (p < 0.05) among months (nd = not determined).

	L (mm)	H (mm)	W (mm)	WWc (g)	WWst (g)	DWst (g)	WWv (g)	DWv (g)
October 2021	62.9 ± 3.3 a (100)	31.9 ± 5.2 abcd (100)	16.1 ± 1.5 a (100)	17.7 ± 3.5 b (100)	6.3 ± 1.2 a (100)	1.6 ± 0.5 a (20)	5.2 ± 1.3 cd (20)	5.2 ± 1.3 ab (20)
November 2021	60.6 ± 3.0 cd (60)	32.7 ± 1.5 abc (60)	15.3 ± 0.7 bc (60)	18.0 ± 2.9 ab (100)	6.0 ± 0.8 ab (20)	1.5 ± 0.2 ab (20)	5.6 ± 1.2 abc (20)	5.3 ± 1.1 ab (20)
December 2021	57.7 ± 2.4 fg (90)	30.8 ± 1.2 d (90)	14.2 ± 0.8 ef (90)	14.6 ± 2.0 d (100)	5.1 ± 0.9 cd (81)	1.2 ± 0.2 cde (20)	4.5 ± 0.5 d (20)	4.2 ± 0.5 c (20)
January 2022	57.6 ± 2.2 g (81)	nd	14.3 ± 0.6 ef (81)	17.0 ± 0.9 b (100)	2.5 ± 0.7 g (81)	1.3 ± 0.2 bcde (20)	nd	4.8 ± 0.6 bc (20)
February 2022	59.1 ± 3.0 def (80)	31.7 ± 1.3 bcd (80)	15.5 ± 2.2 b (80)	11.4 ± 1.9 e (100)	3.1 ± 0.7 f (80)	0.6 ± 0.1 fg (20)	3.4 ± 0.4 e (20)	3.2 ± 0.5 d (20)
March 2022	60.5 ± 3.4 cd (100)	32.1 ± 1.7 abc (100)	14.9 ± 1.0 cd (100)	16.2 ± 2.4 c (100)	3.5 ± 0.7 ef (100)	0.8 ± 0.2 f (20)	5.6 ± 0.8 abc (20)	5.3 ± 0.8 ab (20)
April 2022	59.3 ± 2.5 de (95)	31.7 ± 1.7 cd (95)	15.0 ± 1.6 bcd (95)	16.0 ± 2.0 c (100)	3.7 ± 0.7 e (95)	0.5 ± 0.1 g (20)	5.0 ± 0.5 cd (20)	3.2 ± 0.4 d (20)
May 2022	58.1 ± 2.4 efg (100)	30.9 ± 1.3 d (100)	14.1 ± 0.8 f (100)	15.1 ± 2.3 cd (100)	5.1 ± 0.9 d (100)	1.1 ± 0.2 e (20)	5.2 ± 0.6 bcd (20)	4.8 ± 0.6 bc (20)
June 2022	60.3 ± 3.9 d (100)	32.1 ± 2.5 abc (100)	14.7 ± 0.8 de (100)	17.5 ± 2.8 b (100)	5.6 ± 0.9 b (100)	1.4 ± 0.2 bcd (20)	6.1 ± 1.1 ab (20)	5.8 ± 1.0 a (20)
July 2022	61.6 ± 2.7 abc (100)	32.6 ± 1.6 abc (100)	15.2 ± 1.3 bcd (100)	18.4 ± 3.3 ab (100)	5.5 ± 1.0 bc (100)	1.1 ± 0.2 de (20)	6.1 ± 0.6 ab (20)	5.6 ± 0.5 ab (20)
August 2022	62.1 ± 2.4 ab (100)	32.7 ± 1.5 ab (100)	14.9 ± 0.6 cd (100)	19.0 ± 3.2 a (100)	5.2 ± 0.8 cd (100)	1.1 ± 0.2 de (20)	6.4 ± 1.2 a (20)	6.1 ± 1.2 a (20)
September 2022	61.5 ± 2.7 bc (100)	32.8 ± 1.7 a (100)	14.7 ± 0.9 de (100)	19.3 ± 3.4 a (100)	5.9 ± 1.0 ab (100)	1.4 ± 0.3 bc (20)	5.9 ± 1.0 abc (20)	5.5 ± 1.0 ab (20)

), the mean total length ranged from 57.6 and 62.9 mm, indicating that clams were generally of similar size across sampling dates. Overall, clams sampled between July and October 2021 tended to have greater L, H, and W values, as well as higher WWc. The mean dry weight of the soft tissues (DWst) was significantly higher in October (1.64 g) than in most other sampling months, although it did not differ significantly from that recorded in November 2021 (1.50 g) (Table 2). Mean CI decreased from 27.7–31.3 in October–December 2021 to 14.9 in April 2022 (Figure 1). Thereafter, mean CI increased, reaching significantly higher values in May, June, and September 2022.

Although the differential hemocyte count (DHC) was not significantly different among monthly samples, a lower proportion of hyaline hemocytes (HH) than granular hemocytes (GH) was observed throughout the sampling period (

Table 3. Monthly means (±SD) of differential hemocyte count (DHC; %), including hyaline hemocytes (HH) and granular hemocytes (GH), phagocytic hemocyte count (PHC; %), and protein concentration in the hemolymph (HPC; µg mL⁻¹) of the yellow clam Amarilladesma mactroides sampled from October 2021 to September 2022 on Cassino beach, southern Brazil. Superscript letters indicate significant differences (p < 0.05) among months (nd = not determined).

	DHC		PHC	HPC
	HH	GH
October 2021	37.5 ± 11.0	62.5 ± 11.0	16.7 ± 2.5	92.9 ± 27.2 bcd
November 2021	42.2 ± 10.8	57.8 ± 10.8	16.7 ± 1.4	nd
December 2021	46.3 ± 15.6	53.7 ± 15.6	16.7 ± 1.1	96.20 ± 21.5 abcd
January 2022	43.0 ± 8.3	57.0 ± 8.3	16.7 ± 0.5	151.40 ± 27.2 ab
February 2022	36.2 ± 34.7	63.8 ± 34.7	16.7 ± 1.3	130.45 ± 24.8 abc
March 2022	34.7 ± 14.0	65.3 ± 14.0	16.7 ± 0.3	62.83 ± 1.5 cd
April 2022	38.8 ± 18.4	61.2 ± 18.4	16.6 ± 0.4	101.78 ± 24.3 abcd
May 2022	32.5 ± 22.5	67.5 ± 22.5	16.7 ± 1.9	156.13 ± 21.9 ab
June 2022	15.5 ± 18.0	84.5 ± 18.0	16.7 ± 2.0	42.20 ± 3.1 d
July 2022	41.0 ± 17.8	59.0 ± 17.8	16.7 ± 4.7	243.27 ± 78.1 a
August 2022	36.2 ± 29.0	63.8 ± 29.0	16.7 ± 8.3	113.20 ± 16.3 abc
September 2022	39.8 ± 25.1	60.2 ± 25.1	16.7 ± 8.1	63.13 ± 6.7 cd

). The highest proportion of HH (46.3%) was observed in December 2021 and the lowest (15.5%) in June 2022. No significant differences were found for the phagocytic hemocyte count (PHC), as mean values were identical (16.7%) throughout the sampling period, with the exception of April 2022, when it was 16.6% (Table 3). The mean concentration of total hemolymph proteins (HPC) showed significant differences, varying from 42.2 µg mL⁻¹ in June 2022 to a maximum of 243.3 µg mL⁻¹ in July 2022.

Analyses of the histological sections (Figure 2) indicated that the gonads went through two periods of maturation during the sampling period: from October to December 2021 and from May to July–August 2022. In both periods, polygonal-shaped oocytes were free in the lumen. The gonads of females sampled in January and February 2022 presented immature, mature, and residual oocytes, with peduncular-shaped immature oocytes still attached to the walls, suggesting that these females were in the partial spawning stage. In March and April 2022, based on the presence of immature cells, abundant connective tissue, and no sexual differentiation, the gonads were classified as resting or recovering. Signs of early maturation (presence of mature polygonal oocytes and round oocytes) were observed from May to June 2022. Gonadal development proceeded as more advanced stages of gametogenesis, particularly an increased follicle size, were observed. The oocyte size varied significantly throughout the sampling period (

Table 4. Mean (±SD), minimum, and maximum values of oocyte diameter (µm), number of females sampled (F), and total number of oocytes measured (n) of yellow clam Amarilladesma mactroides collected on Cassino beach, southern Brazil, from October 2021 to September 2022. Superscript letters indicate significant differences (p < 0.05) among months (nd = not determined).

	Mean (± SD)	Minimum–Maximum	F (n)
October 2021	33.58 ± 7.61 bc	14–51	5 (100)
November 2021	38.00 ± 7.21 a	21–58	3 (60)
December 2021	36.00 ± 6.33 a	18–54	5 (100)
January 2022	36.00 ± 5.06 ab	25–53	4 (50)
February 2022	32.07 ± 4.94 cd	20–41	4 (60)
March 2022	nd	nd	4 (0)
April 2022	nd	nd	5 (0)
May 2022	30.15 ± 4.67 e	16–44	5 (100)
June 2022	31.40 ± 6.53 de	15–45	5 (100)
July 2022	31.00 ± 6.26 de	13–50	3 (60)
August 2022	23.83 ± 6.14 f	13–39	5 (100)
September 2022	31.04 ± 5.92 de	16–46	5 (100)

). Because only follicles in the process of formation were observed in March and April 2022, no oocytes were detected.

The smallest oocytes (mean diameter of 23.83 µm) were observed in August 2022 (Table 4), whereas significantly larger oocytes occurred from November 2021 to January 2022, when the mean diameter was ≥36 µm. The maximum diameter of the oocytes varied from 39 to 58 µm (Table 4). The largest oocyte diameter (≥51 µm) was observed from October 2021 to January 2022. From May to September 2022, the maximum oocyte diameter ranged from 39 µm to 50 µm.

The concentrations of crude protein, total lipids, glycogen, and lactate varied significantly over the sampling period (Figure 1;

Table 5. Means (± SD) of crude protein (%), total lipids (%), glycogen (mg g⁻¹) and lactate (mg g⁻¹) of yellow clam Amarilladesma mactroides collected from October 2021 to September 2022 on Cassino beach, southern Brazil. Different superscript letters within columns indicate significant differences (p < 0.05).

	Crude Protein	Total Lipids	Glycogen	Lactate
October 2021	46.91 ± 0.88 f	5.25 ± 0.41 ab	1.13 ± 0.82 fgh	0.29 ± 0.10 de
November 2021	47.27 ± 1.40 f	5.06 ± 0.23 abc	0.73 ± 0.91 h	0.24 ± 0.10 defg
December 2021	50.35 ± 0.32 e	4.25 ± 0.07 cde	1.86 ± 1.11 ef	0.18 ± 0.05 g
January 2022	58.69 ± 0.86 cd	4.42 ± 0.38 bcde	1.58 ± 1.11 efg	0.32 ± 0.11 d
February 2022	66.38 ± 0.48 a	4.13 ± 0.25 de	2.03 ± 0.83 de	0.32 ± 0.19 de
March 2022	64.04 ± 0.66 a	3.73 ± 0.35 e	2.44 ± 0.49 de	0.75 ± 0.11 ab
April 2022	61.23 ± 0.00 b	3.91 ± 0.30 e	0.92 ± 0.18 gh	0.22 ± 0.28 fg
May 2022	57.49 ± 1.13 d	5.18 ± 0.24 ab	3.40 ± 0.38 bc	0.96 ± 0.16 a
June 2022	60.70 ± 1.10 bc	4.09 ± 0.26 de	3.44 ± 0.50 bc	0.59 ± 0.29 bc
July 2022	59.43 ± 0.16 bcd	3.67 ± 0.29 e	5.45 ± 0.60 ab	0.24 ± 0.15 efg
August 2022	65.05 ± 0.59 a	4.78 ± 0.06 abcd	2.88 ± 0.44 bc	0.40 ± 0.25 cd
September 2022	60.66 ± 0.25 bc	5.44 ± 0.36 a	7.57 ± 0.78 a	0.35 ± 0.25 def

). The mean crude protein content increased significantly from 46.91–47.27% in October–November 2021 to 64.04–66.38% in February–March 2022. Protein content then declined significantly in April and May 2022 before reaching a second peak of 65.05% in August 2022 (Table 5). Changes in total lipid content were inversely related to those of crude protein (Table 5). Total lipid concentrations ranged from 3.67% in July 2022 to 5.44% in September 2022 (Figure 1). Lipid content decreased significantly between October 2021 and March–April 2022, increased in May, declined again in June–July 2022, and returned to relatively high levels in September 2022. Hemolymph glycogen concentrations exhibited marked temporal variation, with an approximately tenfold difference over the sampling period (Table 5). The mean glycogen concentration increased from a minimum of 0.73 mg g⁻¹ in November 2021 to a maximum of 7.57 mg g⁻¹ in September 2022. Following a gradual increase from November 2021 to March 2022, the glycogen concentration declined significantly from 2.44 mg g⁻¹ in March to 0.92 mg g⁻¹ in April. Concentrations then increased steadily, reaching 5.45 mg g⁻¹ in July 2022, decreased to 2.88 mg g⁻¹ in August 2022, and peaked at 7.57 mg g⁻¹ in September 2022. Lactate concentrations also varied significantly throughout the sampling period (Table 5). The highest concentration was recorded in May 2022 (0.96 mg g⁻¹), whereas the lowest was observed in December 2021 (0.18 mg g⁻¹). Overall, the mean lactate concentrations remained between 0.18 to 0.40 mg g⁻¹, except in March, May and June 2022, when they increased to 0.75, 0.96 and 0.59 mg g⁻¹, respectively.

The fatty acid composition of soft tissues is presented in

Table 6. Principal fatty acid content (mg g⁻¹ of total lipids) of the soft tissues of the yellow clam (Amarilladesma mactroides) sampled monthly from October 2021 to September 2022 on Cassino beach, southern Brazil. Within rows, superscript letters represent significant differences (p < 0.05). Sample size is presented in brackets as (n).

	October 2021 (3)	November 2021 (3)	December 2021 (3)	January 2022 (2)	February 2022 (3)	March 2022 (2)	April 2022 (2)	May 2022 (3)	June 2022 (3)	July 2022 (3)	August 2022 (3)	September 2022 (4)	Pooled SEM 1	p
14:0	31.3 cdef	23.6 f	26.5 def	37.5 bcdef	60.9 a	30.4 def	26.3 ef	32.0 cdef	52.2 ab	41.2 bcd	45.8 bc	37.6 cde	2.69	0.016
16:0	168.3 ab	158.7 ab	150.5 ab	153.8 ab	151.4 ab	143.0 ab	125.7 b	146.8 ab	149.7 ab	152.0 ab	166.0 ab	188.3 a	9.56	0.020
16:1n-7	38.6 a	14.0 b	9.6 b	15.1 ab	13.7 b	14.1 b	15.4 ab	10.1 b	29.1 ab	24.6 ab	12.8 b	22.5 ab	3.73	0.009
18:0	61.5 ab	78.0 a	80.4 ab	65.6 ab	55.3 ab	47.6 b	61.9 ab	52.9 ab	54.2 ab	53.4 ab	57.9 ab	68.8 ab	5.30	0.024
18:1n-9	21.8 ab	23.1 a	23.3 ab	13.7 ab	8.5 b	14.8 ab	13.2 ab	13.0 ab	12.9 ab	14.6 ab	10.8 ab	19.0 ab	2.84	0.019
18:1n-7	27.8 a	21.9 ab	18.8 abcd	14.7 bcd	10.8 d	15.3 bcd	11.6 cd	10.1 d	17.9 abcd	17.0 abcd	12.0 bcd	21.0 abc	3.41	0.034
18:2n-6	2.0	2.1	2.6	2.8	2.4	3.8	2.9	3.2	3.0	2.3	1.8	2.4	0.54	0.488
18:3n-4	10.8 ab	10.1 a	11.4 a	7.3 abc	2.2 c	2.8 bc	2.5 c	2.9 bc	1.8 c	9.2 ab	6.8 abc	8.2 abc	1.27	0.005
18:3n-3	20.3 abcd	23.9 ab	25.9 a	22.4 abc	11.0 e	15.7 cde	16.7 bcde	13.2 de	14.0 de	15.1 cde	12.8 de	16.5 bcde	2.62	0.031
20:5n-3	33.1 ab	18.4 abc	18.7 abc	17.9 abc	14.6 bc	27.2 ab	28.5 ab	22.3 ab	35.7 a	21.4 abc	12.7 c	31.8 ab	6.45	0.027
22:5n-3	16.9	12.2	10.4	7.9	5.6	17.2	15.9	11.5	14.1	9.0	8.8	16.5	4.33	0.520
22:6n-3	30.0 a	19.8 ab	12.9 ab	10.8 ab	5.7 b	27.3 a	26.3 a	17.6 ab	18.6 ab	11.9 ab	12.2 ab	28.6 a	6.57	0.017
SFA 2	282.1 abc	282.2 abc	280.7 abc	276.1 abc	284.2 abc	237.5 c	239.4 c	250.1 bc	270.4 bc	264.4 bc	290.2 ab	318.6 a	16.65	0.023
MUFA 3	118.0 a	88.3 ab	81.0 abc	63.8 bc	44.5 c	63.8 bc	63.7 bc	52.5 bc	78.8 abc	74.6 bc	53.5 bc	84.8 ab	13.02	0.018
n-6 PUFA 4	4.5 b	6.7 ab	5.3 ab	7.3 ab	5.0 b	6.7 ab	5.9 ab	8.0 a	7.1 ab	6.3 ab	5.3 b	5.9 ab	0.91	0.035
n-3 HUFA 5	111.5 a	85.0 ab	76.5 ab	66.1 ab	41.6 b	98.4 ab	96.8 ab	72.0 ab	89.8 ab	64.4 ab	53.2 ab	102.1 ab	21.12	0.048
DHA/EPA	0.9 ab	1.1 a	0.7 bcd	0.6 cde	0.4 e	1.0 a	0.9 ab	0.8 bc	0.5 de	0.6 cde	0.9 ab	0.8 bc	0.07	0.013
n-3/n-6	25.2 a	13.1 ab	14.5 ab	9.4 ab	9.3 b	15.4 ab	19.4 ab	9.8 ab	12.7 ab	10.5 ab	10.6 ab	22.3ab	4.81	0.048

¹ Pooled standard error of means; ² SFA = Σ saturated fatty acids; ³ MUFA = Σ monounsaturated fatty acids; ⁴ n-6 PUFA = Σ n-6 ≥ 18:2n-6; n-3 HUFA = ⁵ Σ n-3 ≥ 18:3n-3.

. Regardless of the sampling period, the predominant fatty acids were myristic (14:0), palmitic (16:0), stearic (18:0), and eicosapentaenoic (20:5n-3; EPA) acids, whereas palmitoleic (16:1n-7), oleic (18:1n-9), vaccenic (18:1n-7), linolenic (18:3n-3), and docosahexaenoic (22:6n-3; DHA) acids were present in intermediate levels. In general, there was a predominance of saturated fatty acids over monounsaturated fatty acids. Likewise, the levels of n-3 polyunsaturated fatty acids (n-3 PUFA) were much higher than n-6 PUFA levels, resulting in n-3/n-6 ratios generally greater than or close to 10. Among n-3 PUFA, the EPA content was higher than that of DHA; therefore, the DHA/EPA ratios were consistently lower than 1.0 (except in February 2022). While significant variations in the concentrations of most fatty acids were observed throughout the sampling period, the levels of linoleic acid (18:2n-6) were not significantly different (Table 6). The concentration of n-3 PUFA showed a significant decrease from October 2021 (111.5 mg g⁻¹) to February 2022 (41.6 mg g⁻¹), which, in a way, reflects the also significantly lower concentrations of DHA. Such reductions occurred simultaneously with the most intense spawning period, as previously indicated (October to December) (Figure 3). A similar decrease in the concentration of n-3 PUFA can also be observed from March–April (96.8–98.4 mg g⁻¹) to August (53.2 mg g⁻¹), when the less intense spawning peak was estimated to occur from May to July 2022.

4. Discussion

Environmental variables, including temperature, salinity, dissolved oxygen, and pH, varied considerably throughout the 12-month sampling period, coinciding with significant temporal variation in several biometric, physiological, reproductive and biochemical parameters. Significant differences were observed in the biometric characteristics of the clams, as well as in the contents of crude protein, total lipids, fatty acids, glycogen, and lactate, gonadal histology (including oocyte diameter), and hemolymph protein concentration. In contrast, no significant temporal variation was detected in differential hemocyte counts or the proportion of phagocytic hemocytes, suggesting that these immune parameters may be less responsive to seasonal changes under the environmental conditions experienced during the study. Fluctuations in environmental conditions, particularly salinity, may affect hemolymph composition through the regulation of amino acids and ions [26,27]. Interestingly, the lowest HPC (42.2 µg mL⁻¹) was recorded in June 2022, coinciding with the lowest salinity (28) observed during the study. Similarly, significant reductions in HPC have been reported in the oyster Crassostrea madrasensis exposed to salinities below and above its optimal range [28]. Although the present study cannot establish a causal relationship between salinity and HPC, the concomitant decrease in both variables suggests that changes in salinity may have contributed to the observed variation in hemolymph protein concentration.

The proportion of granular hemocytes (GH) was consistently higher than that of hyaline hemocytes, a pattern that has been associated with bivalves exposed to stressful conditions [29]. Granular hemocytes play an important role in cellular defense, including the phagocytosis and elimination of pathogens and foreign materials [30]. However, the absence of significant temporal variation in differential hemocyte counts and phagocytic hemocyte indicates that the predominance of GH did not necessarily reflect a compromised immune condition. Rather, our results are consistent with the previously described immunological status for A. mactroides collected during different seasons along the southern coast of Brazil [31]. Taken together, the relatively stable hemocyte profile and phagocytic activity suggest that the clams remained in good general health throughout the study period. At the same time, the seasonal variation observed in the biochemical and reproductive parameters indicates pronounced temporal changes in the physiological and reproductive condition of A. mactroides along the southernmost coast of Brazil, consistent with previous studies conducted in other regions within the species’ natural distribution [13,15,32,33].

Based on gonad histology, oocyte diameter, condition index, total lipid content, and n-3 PUFA levels, we identified two reproductive peaks over the 12-month study: a major peak in spring–summer (October–December 2021) and a less pronounced peak during autumn–winter (May–July 2022). The occurrence of multiple reproductive events appears to be consistent with patterns reported for other A. mactroides populations, although the timing and intensity of these events vary geographically. Along the Argentinean coast, spawning has been reported from late November to mid-March [1], while two distinct reproductive periods have also been described, with a major peak in spring and a less intense peak in summer [12]. Similarly, two annual reproductive events have been reported for A. mactroides in Argentina, occurring in winter and spring [13] and in spring and early summer [32]. On the Uruguayan coast, spawning individuals were observed throughout the year, but with two more pronounced peaks in December and August and three less intense peaks in October, March, and May [33]. Similarly, on the coast of Santa Catarina, Brazil, spawning occurred throughout the year but was more pronounced during autumn and winter [15]. Such differences in the timing and intensity of maturation and spawning among geographic populations are common in bivalves and may reflect differences in local environmental conditions [14,34,35]. For example, similar geographical variation has been reported in the congener Mesodesma donacium, for which two reproductive peaks in spring–summer and autumn were observed on the northern coast of Chile [36], whereas only one spawning period was recorded in a population from the southern coast of the country [37]. These differences have been attributed to variation in environmental conditions among locations. Accordingly, the differences in reproductive timing observed among A. mactroides populations along the coasts of Uruguay, Argentina, and Brazil may be related to geographical and latitudinal variation, which can influence environmental conditions, particularly temperature and salinity, and food availability [13,37,38,39,40]. Thus, the environmental conditions that favor gametogenic development and spawning in A. mactroides may occur at different times of the year depending on geographic location and latitude, resulting in spatial variation in the timing and intensity of reproductive events.

Seasonal changes in temperature typically stimulate gametogenesis [13], whereas abrupt temperature changes can trigger spawning [41]. Our results indicate that yellow clams in the southernmost region of Brazil entered a reproductive resting phase during autumn, particularly in March and April 2022. Histological examination of clams sampled during these months revealed little evidence of gonadal differentiation, with only residual oocytes, very small follicles, and large interfollicular spaces observed. Consequently, oocyte measurements were not possible during this period. A similar condition was reported for A. mactroides [33], with the authors also noting the difficulty of distinguishing between sexes during the resting phase. Likewise, following complete spawning, the resting period was characterized by the recovery and reorganization of follicular tissue, with sex identification possible only due to the presence of residual oocytes [32]. In A. mactroides from the Argentinean coast, the reproductive resting phase occurred from January to March–April, and oocyte measurements were likewise not possible during this period [13]. These similarities suggest that the reproductive resting phase observed in southern Brazil is consistent with the seasonal reproductive pattern previously described for this species across its distributional range.

The reproductive resting phase identified in this study (March–April 2022) followed an abrupt decrease in seawater temperature. In February 2022, seawater temperature reached 28.2 °C, decreasing to 19.5 °C in March, a decrease of 8.7 °C that represented the largest temperature change recorded during the study period. This temporal association raises the possibility that a sudden decrease in temperature may contribute to the onset of the reproductive resting phase in A. mactroides. However, this hypothesis requires further investigation under controlled conditions. A significant association between temperature fluctuations and gonadal development in A. mactroides has been reported [13], although the authors found that oocyte maturation and maximum size during winter (July–August) were associated with the lowest temperatures. Thus, while low temperatures may favor oocyte development during certain stages of the reproductive cycle, an abrupt temperature decrease may have a different physiological effect, potentially contributing to the transition from active reproduction to the resting phase.

Oocyte diameter is widely regarded as a useful indicator of the reproductive cycle in bivalves because it closely reflects the stage of gametogenic development and is influenced by environmental conditions, age, and life-history strategy [42,43,44]. As in other bivalves, the largest oocytes are generally observed in mature, spawning or partially spawned individuals [43,45]. In the present study, the oocyte diameter in A. mactroides ranged from 13 µm to 58 µm, which is consistent with values reported in previous studies of this species. Oocyte diameters ranging from 17 µm to 45 µm have been reported depending on the degree of maturation [33], while three stages of maturation have been distinguished based on oocyte size: incipient maturation, with oocytes generally ranging from 10 to 25 µm; advanced maturation stage, with oocytes > 35 µm; and full maturation, with oocytes > 45 µm [32]. Mature oocytes ready to spawn have been reported to range from approximately 50 to 55 µm [1,13], whereas newly fertilized eggs have been reported to average 51.2 µm in diameter [46]. A reproductive scale based on oocyte size has been proposed for assessing gametogenic development in bivalves [44], with a minimum diameter of 45–50 µm suggested as indicative of maturity in A. mactroides [17]. The maximum oocyte diameter observed in the present study (58 µm) falls within the range previously reported for spawning females and, together with the observed seasonal changes in oocyte size, supports the use of oocyte diameter as a reliable indicator of reproductive development in A. mactroides. Our findings, therefore, provide further support for a minimum oocyte diameter of 45 µm as a useful threshold for identifying mature individuals in this species.

The condition index (CI) is a useful indicator for assessing the reproductive cycle of bivalves. In the present study, significantly higher CI values (≥25) were recorded when histological analyses indicated that the gonads were mature, corresponding to the most intense reproductive period (October–December 2021). Subsequently, CI values declined to means below 20 in February 2022 and remained low during the reproductive resting phase in March and April 2022, consistent with the patterns observed in gonadal histology and oocyte diameter. Similar relationships between CI and reproductive activity have been reported for several bivalve species [40,47,48,49,50]. From April 2022 onward, following the end of the gonadal resting phase, CI increased significantly, suggesting the resumption of gametogenesis and preparation for a subsequent reproductive period. Overall, the temporal pattern of CI closely mirrored the seasonal changes in gonadal development, supporting its use as a complementary, non-histological indicator of reproductive condition in A. mactroides.

Total lipid content was also a useful indicator for characterizing the reproductive cycle of A. mactroides. Significantly higher lipid levels were observed prior to the reproductive peaks, followed by a marked decrease in March and April, coinciding with the reproductive resting phase and the lowest CI values. Similar seasonal lipid depletion has been reported for the Pacific oyster Crassostrea gigas, in which lower lipid concentrations were observed during the spawning period, suggesting that lipid reserves are mobilized during the final stages of gametogenesis and spawning [47,51]. A similar process may have occurred in A. mactroides, particularly from February to April 2022, when low lipid levels coincided with reduced CI values. The accumulation of lipids prior to periods of intense reproductive activity suggests that these reserves may be mobilized to support gametogenesis and gonadal development [47,52,53,54], as lipids play important roles in the formation and maturation of gonads and gametes [55,56]. Consistent with this interpretation, an increase in lipid content before spawning followed by a decrease immediately afterward has been reported in Arca noae [56]. Similarly, increased lipid reserves associated with gametogenesis have been documented in other burrowing bivalves, including Donax trunculus and Tapes philippinarum [57,58]. In contrast, higher total lipid concentrations in the sand-dwelling bivalve Anomalocardia brasiliana were observed during autumn and were interpreted as the accumulation of energy reserves for subsequent gametogenesis [59]. These contrasting patterns suggest that seasonal lipid dynamics may vary among species and may reflect differences in reproductive strategies, environmental conditions, and food availability. Thus, in A. mactroides, the temporal pattern observed in the present study is consistent with the mobilization of lipid reserves during periods of intense reproductive activity, followed by replenishment during subsequent phases of the reproductive cycle.

In bivalves, the accumulation of lipids, particularly of fatty acids, during gametogenic development is essential for reproduction and early larval development [47,52,54,55,56]. In addition to serving as metabolic substrates and major energy reserves, specific fatty acids are essential for maintaining cell membrane structure and function [60,61]. Accordingly, oocyte maturation is generally accompanied by the accumulation of fatty acids. Saturated and monounsaturated fatty acids are preferentially catabolized as energy substrates, whereas n-3 PUFAs, particularly DHA, are selectively retained, reflecting their important structural and physiological functions [62]. DHA is a key component of cellular membranes and plays important roles in the structural and functional processes associated with oogenesis and embryogenesis across a wide range of taxa, including mammals [63], fish [64], crustaceans [65], and bivalves [62]. In the present study, we observed significant reductions not only in total lipid concentration in the soft tissues but also in n-3 HUFAs and, to a lesser extent, DHA. Notably, these reductions coincided with the two spawning peaks identified in the present study: the more pronounced reproductive peak, extending from October 2021 to February 2022, and the less intense peak observed from May to July 2022. This temporal correspondence suggests that the depletion of lipid reserves, particularly n-3 HUFA and DHA, may be associated with their mobilization and transfer to gametes during spawning, supporting the energetic and structural requirements of reproduction and early development.

5. Conclusions

Our study demonstrated that the yellow clam, A. mactroides, exhibits two annual reproductive peaks in the southernmost region of Brazil: a more pronounced peak during spring–summer (October–December) and a less intense peak in autumn–winter (May–July). Oocyte diameter, condition index, total lipid content, and n-3 PUFA levels of the soft tissues proved to be useful complementary indicators for characterizing and identifying the reproductive peaks of this ecologically and socioeconomically important bivalve.