Donax striatus were collected on Ajuruteua Beach, northeast Pará (00°49’57”S; 46°36’01”W) (Fig. 1), with a rainy season between December and May, a dry season between June and November, and a regime of semidiurnal macrotides (4-6 m). The beach showed dissipative characteristics, with 200 to 300 m low tides, sediment with a fine grain size, and strong energy waves (Martorano et al. 1993, Alves and El-Robrini 2006).

Sampling was performed monthly during the syzygy low tide from March 2015 to April 2016, and 30 individuals were randomly collected along the beach. Subsequently, each individual was measured for shell length (SL) with a caliper and weighed for total weight and soft parts wet weight using a digital balance to 0.01 g. To convert the soft parts dry weight to ash-free dry mass (AFDM), the specimens were dried for 12 h at 90°C then subjected to ignition in a muffle oven at 550°C for 24 h (Brey 1986).

Simultaneously, during the sampling surveys the environmental variables at the beach were measured, including sea surface temperature (SST), with a digital thermometer with a precision of 0.1°C, and salinity, with a portable refractometer with a scale of 1/100. The National Meteorology Institute provided precipitation data. The sediment organic matter was analysed through the ignition methodology proposed by Dean (1974). Seawater was collected at the beach using a polyethylene bottle and refrigerated for further analysis of chlorophyll a following the methodology of Cetesb (2014).

Histological analysis and condition index

For the histological analysis, the visceral mass was removed from the shell and immersed in a salty Davidson solution for 24 h. Subsequently the gills, labial palps, and mantle border were also removed and the gonads were transferred to 70% alcohol. In the laboratory, the modified histological method of Junqueira and Junqueira (1983) was used. Transverse 5 μm sections were sliced using a manual microtome. Gonad sections of D. striatus were examined using a binocular microscope; five stages of gonadal development were observed, and were identified and characterized based on the morphological and cytological criteria described by Narchi (1976) for Anomalocardia fluxuosa and Herrmann et al. (2009) for D. hanleyanus. Photomicrographs of histological sections were captured using Motic Image Plus 2.0, to determine the oocyte number and average oocyte diameter following the methodology described by Herrmann et al. (2009), using Image Tool 3.0 software.

According to Boehs et al. (2008), the condition index (CI) could provide information related to the processes of glycogen conversion in gametes, sexual maturation and gamete release, using the following formula:

where CI is the condition index, SPWW is the soft parts wet weight and TWW is the total wet weight.

The size at which 50% of the individuals in the population are sexually mature (adults) was estimated from the Galton ogive, and from the proportion of mature males and females (stage II, maturation; stage III, Ripe; stage IV, spawning; and stage V, cytolysis) in different size classes, using the logistic equation according to McCullagh and Nelder (1989):

where SL₅₀ is the proportion of females and males with mature gonads in each size class (SL) and a, b and x₀ are parameters.

The size at first maturity was estimated by plotting the frequency of maturation (Y) against the size class (X), generating a sigmoid, where an abscissa was traced at the point corresponding to 50% until it intersects the curve. This meeting point corresponds in the x-axis to the size at first maturity. The above equation was adjusted by non-linear least squares using the quantum Newton algorithm of the SigmaPlot software package, version 11 (2008).

Statistical analyses

The assumptions of normality and homoscedasticity of the environmental and biological data were tested by Kolmogorov-Smirnov and Levene tests, respectively. All results were considered statistically significant at α≤0.05. From the analysed variables, only oocyte abundance, ripe and spawning stages met these assumptions, so the t-test was carried out to verify differences between the dry and wet seasons.

The nonparametric Mann-Whitney (U) test was applied when the assumptions of parametric tests were not met with either raw or transformed data. Therefore, this test was employed on AFDM, CI, oocyte diameter and environmental variables also to compare mean values between seasons.

The generalized linear model (GLM) was used to examine the relationships between CI, biomass, oocyte abundance, ripe and spawning stages and the explanatory variables (SST, salinity, precipitation, chlorophyll a and sediment organic matter). The GLM was constructed using a Gaussian model identity link function. The Akaike information criterion (AIC) (Akaike 1973) was used to compare the models, with lower values indicating a more parsimonious model than a model fit with a higher AIC.

The population sex ratio was compared between seasons using the chi-square test (χ²). The t, Mann-Whitney and chi-square tests were performed with the PAST software, version 3.02 (Hammer et al. 2001), while the GLM was carried out using the open R software, version 3.4.3 (R Core Team 2017).

During the study period, when there was no rainfall (September to December 2015), the SST at the beach reached a maximum of 36°C (October 2015), the salinity was 36 (September 2015), and chlorophyll a reached 3.7 mg m^–3 (December 2015); however, the organic matter peaked during the rainy season in January 2016 at 2.6% (Fig. 2). The organic matter was significantly higher in the rainy season (U=0.0023; p<0.05), and SST, chlorophyll a and salinity were not significantly different between seasons (U=0.4954, U=0.3518 and U=0.1293, respectively; p>0.05).

During the study period, 420 individuals were analysed, of which 207 were females (52%) and 193 (48%) were males, with their size range varying from 2.8 mm to 39.1 mm SL. There was no significant difference in the sex ratio of the samples (p>0.05), which showed a balanced proportion of 1 M: 1 F (χ²=0.49; p>0.05). Monthly there were no differences in the sex ratio, except in April 2016, when females (67%) were significantly more abundant than males (33%) (χ²=3.33; p<0.05).

Based on the microscopic images of the gonadal tissues, five stages of gonadal development of males and females of D. striatus were assigned and described: I, immature; II, maturation; III, ripe; IV, spawning; and V, cytolysis.

In females, the immature stage was characterized by ovarian follicle formations, with clusters of mother cells adhering to the follicular wall, and a low frequency of basophil oocytes (Fig. 3A). The maturation stage showed different phases of germinative cells, with the predominance of basophils and pre-vitellogenic and vitellogenic oocytes, while mature cells had lower frequency; however, the ovarian follicles were well delimited, with thin walls (Fig. 3B). The ripe stage was characterized by different phases of germinative cells, but there was a high frequency of mature oocytes and the follicles had relatively large diameters, with the follicle wall distended, and a considerable quantity of oocytes within the follicles (Fig. 3C).

In the spawning stage the follicular wall had interfollicular tissue, and fewer cells in pre-vitellogenic, vitellogenic and mature stages were observed, leaving spaces within the ovarian follicles (Fig. 3D). The cytolysis stage was characterized by a thick follicular wall, and the ovarian follicles were empty or with residuals cells, showing a proliferation of new cells (Fig. 3E).

In males, the immature stage was characterized by the presence of only mother cells and the beginning of testicular tube formation, and gametes in the spermatogonia and spermatocyte stages (Fig. 3F). In the maturation stage, the testicular tubes were already fully formed, with the presence of germ cells in spermatogonia, spermatid and spermatocyte phases. The number of spermatozoa, when present, were reduced and dispersed in the testicular lumen (Fig. 3G). In the ripe stage, there were increased gametogenic processes, including all phases of germ cells, with a predominance of spermatozoa in the centre of the testicular tubes. The testicular tubes had a relatively large diameter with limited internal space (Fig. 3H).

In the spawning stage, the spermatozoa were expelled, leaving empty spaces in the testicular tubes. The tube diameter was reduced and no follicular wall was observed (Fig. 3I). In the cytolysis stage, most spermatozoa had been expelled from the testicular tubes; interfollicular tissue with stem cells or spermatogonia was observed, and the walls of tubules were considerably reduced (Fig. 3J).

Seasonal variation of condition index and biomass

The monthly mean CI ranged from 6.9% to 15.5% (11.1±4.7) and AFDM ranging from 0.005 to 0.009 g (0.0065±0.0031) (Fig. 4B, C). According to the GLM, the CI only had a significant relationship with the sediment organic matter (t value=2.935, p=0.0188) (Table 1). However, the AFDM showed a slight increase in the dry season, being significantly higher than in the rainy season (U=0.056, p<0.05), which may have occurred due to a higher frequency of mature individuals. The CI showed no difference between seasons (U=0.2468, p>0.05), confirming that the reproduction was not seasonal.

In the histological analysis, the presence of distinct stages of gonadal development was observed in most months, so the reproductive cycle of D. striatus could be characterized as continuous. Both sexes showed the spawning stage throughout the study period, indicating continuous spawning.

Higher proportions of mature females (August and November 2015) and mature males (July to October 2015) occurred during the dry season. Higher occurrences of spawning stages occurred in females in May and September 2015 and January and February 2016, and in males between March and May 2015 and December 2015 and February 2016. Therefore, a higher incidence of spawned individuals was observed in the rainy season. Immature phases were more frequent in females in October to November 2015 and in males in May 2015 (Fig. 5). Therefore, the probable recruitment of the species occurred in these periods.

During the study period, the mean oocyte diameter in distinct stages of gonadal development were 20.8, 21.2, 23.2, 19.8, and 21.6 µm for the immature, maturation, ripe, spawning and cytolysis stages, respectively (Table 2). There was no significant difference between seasons (U=0.64179; p>0.05). It was observed during the study period that the maturation stage in all the germinative cell lines shows continuous maturation, revealing a reproductive strategy throughout the study period that has no direct correlation with the environmental variables.

Oocyte diameters varied from 3 to 66 µm, with the histograms showing that the size intervals with highest frequencies were 18 to 23 and 23 to 28 µm, with no clear reduction from these sizes throughout the study period (Fig. 6).

The highest and lowest oocyte abundance occurred in October 2015 and April 2016, respectively (Fig. 4A). The abundance of oocytes showed no significant relationships with environmental parameters (Table 1), and the t-test revealed that oocyte number was not affected seasonally, further confirming a continuous reproductive cycle (t=0.0538; p>0.05).

According to the method of the logistic equation of the Galton ogive (McCullagh and Nelder 1989), sexual maturity occurred in females at 8.9 mm length and in males at 10.9 mm length. When the sexes are grouped, it is estimated that the population will become reproductively active at 12.5 mm length. The second method used to calculate the first sexual maturity size, the logistic curve, confirmed that D. striatus begins to mature between 10 and 15 mm length (Fig. 7).