MATERIAL AND METHODS

Study area. Ubatuba is located in the northern coast of São Paulo State, Brazil. This region has a unique geological conformation and is known for its very irregular coast (^{Ab’Saber, 1955}). Ubatuba is influenced by three water masses: Coastal Water (CW: temperature ≥ 20°C; salinity ≤ 36), Tropical Water (TW: temperature ≥ 20°C; salinity ≥ 36), and South Atlantic Central Water (SACW: temperature ≤ 18°C; salinity ≤ 36) (^{Castro-Filho et al., 1987}). During late spring and early summer, the SACW penetrates into the coastʼs bottom layer and forms a thermocline over the inner shelf at depths of 10 - 15 m (^{Castro-Filho et al., 1987}). During winter, the SACW retreats to the shelf break and is replaced by the CW, resulting in the absence of temperature stratification over the inner shelf during winter (^{Pires, 1992}).

Since October 8, 2008, UBM, UBA and MV are parts of a Marine Protection Area (MPA) (APA Marinha do Litoral- Cunhambebe Sector) created by the Ministry of Environment (decree number 53.525). This MPA was established to ensure the conservation and sustainable use of marine resources. Fishing is only permitted for the subsistence of traditional communities, by amateurs, and as a leisure activity, thus, commercial fishing is not allowed. These restrictions aim to protect the area and promote the rational use of its natural resources, ensuring the regionʼs sustainable development.

Sampling. We captured the swimming crabs monthly from January 1998 through December 1999. In each bay, six sampling stations were established: three stations were located in areas sheltered from the waves (5, 7.5 and 10 m deep), and three were located in exposed areas (10, 15 and 20 m deep) (Fig. 1). The stations (except the 7.5 and 10 m depths) were positioned along transects set parallel to the coastline. These stations were selected according to the following characteristics: their position relative to the bayʼs mouth, the presence of rocky shores or beaches along the bayʼs perimeter, freshwater inflow, proximity to offshore water, depth, and sediment texture. Trawling was conducted on a commercial shrimp fishing boat outfitted with double-rig nets. Each area (18 sites) was trawled monthly (24 months) for 30 min and covered a total of 18,000 m² per trawl.

Fig. 1. Map of the Ubatuba region, in the northeastern coast of São Paulo state, Brazil, showing the three bays (Ubatumirim, Ubatuba and Mar Virado), and their respective sampling stations. Font: adapted from ^{Andrade et al. (2014}). 

At each sampling occasion, the following parameters were recorded: bottom (BT) and surface (ST) water temperature (°C), bottom salinity (BS), depth (m), and percentage organic matter. Water samples were taken with a Nansen bottle to obtain salinity and temperature with an optical refractometer and a thermometer, respectively. Depth was measured with an echobathymeter coupled to a Global Positioning System (GPS). Sediment samples for the determination of organic matter content were taken with a Van Veen grab. Immediately after collection, we put the sediment samples into labelled plastic bags and froze them to minimize the organic matter decomposition until further analyses.

To determine the sediment organic matter content, we put 10 g subsamples in porcelain containers, previously labelled and weighed. They were oven-dried (500°C for 3 hours) and weighed. The difference between the initial and final weight indicated the organic matter content of each sampling station, what was later converted into percentages (^{Heiri et al., 2001}).

Biological data. Achelous spinimanus individuals were identified according to ^{Melo (1996}) and sexed upon inspecting the abdominal morphology (triangular in males and rounded in females) and pleopod number (two pairs in males and four pairs in females). Individuals were categorized into demographic groups: adult males, non-ovigerous adult females, ovigerous females, and juveniles (with the abdomen adhering to the thoracic esternites). Four stages of gonadal development were established for males and females based on the gonad’s size, shape, and color: 1) IM: undifferentiated gonads; 2) RU: rudimentary (males: uncolored filamentous vas deferens; females: whitish, thin filamentous ovary); 3) ED: developing (males: white gonads of small volume, being smaller than the hepatopancreas; females: beginning of maturation, small yellow ovaries); 4) DE: developed (males: largest gonadal development size, white gonads; females: bright yellow ovaries filling almost all thoracic cavity) (^{Santos & Negreiros-Fransozo, 1999}).

Data analyses. A contingency table with the abundance of demographic groups per season was created using a correspondence analysis (CA). To allow a clear visualization of the correlation/abundance of each demographic group per bathymetric zone and season, each datapoint was plotted proportionally to the abundance in the contingency table. A Pearson’s chi-square test with a simulated p-value was used to test the significance of the correlations (based on 2,000 simulations) (^{Nenadic & Greenacre, 2007}).

The reproductive period was estimated based on the monthly frequency of ovigerous females in relation to the total number of females. Similarly, the recruitment was expressed by the monthly frequency of juveniles in relation to the total number of individuals (^{Nenadic & Greenacre, 2007}).

The relationship between the abundance of ovigerous females and juveniles and the environmental parameters (temperature, bottom salinity, and mean organic matter content) was evaluated with a Redundancy Analysis (RDA). The RDA is a multivariate statistical test that measures how strong is the association between groups of variables. The environmental parameters were included in the first group, and the abundance of ovigerous females (OF) and juveniles (J), in the second group. The RDA generates final coordination scores indicating the linear relationship between explanatory and response variables. The RDA produces final coordination scores that summarize the linear relationship between the explanatory and response variables. Only environmental variables with scores higher than 0.4 and lower than - 0.4 were considered as biologically significant (^{Rakocinski et al., 1996}). The CA and RDA were performed using the “ca” and “Vegan” packages (^{Oksanen et al., 2013}; ^{R Development Core Team, 2013}).

RESULTS

The largest differences between surface and bottom temperatures occurred in summer and spring in both years (Fig. 2). In deeper sites (10, 15, and 20 m) there was a large difference between the mean bottom and surface temperatures (thermocline) (Fig. 2). Three water masses were recorded during our survey, which are typical of Ubatuba: Continental Waters (CA; temperature ≥ 20°C, salinity ≤ 36), South Atlantic Central Waters (SACW; temperature ≤ 18°C, salinity ≤ 36), and Tropical Waters (TW; temperature ≥ 20°C, salinity ≥ 36) (Fig. 3). The sites near the coast had the highest organic matter content, whereas the site 20 m deep had the lowest (3.3%).

Fig. 2. Bottom and surface temperature (BT and ST, respectively), seasonal variation in each sampling stations and areas (Shel = sheltered and Exp = exposed) from 1998 to 1999.  

Fig. 3. Diagram showing the seasonal variation of water temperature and salinity from January 1998 to December 1999, in three bays (Ubatumirim, Ubatuba and Mar Virado), from the São Paulo state, southeastern coast of Brazil (CW, Coastal Water; TW, Tropical Water; SACW, South Atlantic Central Water). 

In total, 1,911 individuals of A. spinimanus were captured: 350 adult males, 475 non-ovigerous adult females, 584 adult ovigerous females, and 502 immature individuals. Most individuals, either juveniles or adults, were found at the 7.5 m of depth (Tab. I). However, only adults (males and females, mainly ovigerous) had a high association with deeper areas, especially at 20 m (Fig. 4).

Fig. 4. Achelous spinimanus (Latreille, 1819). Correspondence Analysis (CA) of the abundance of demographic groups in the sampling stations sampled in the Ubatuba region. At the bottom the statistical summary of Pearson’s chi-squared test for randomness of the observed association (J, juveniles; AM, adult males; AF, adult females; OF, ovigerous females; exp, exposed; shel, sheltered). 

Both the reproductive and recruitment periods were continuous (Fig. 5). Males and females with developed gonads were found in all seasons, indicating an intense and constant reproductive activity of both sexes. Even though all demographic groups were found during the entire survey, the temporal distribution, based on the CA analysis (Fig. 6), indicated a relationship between ovigerous females and spring, and between immature individuals and summer and autumn.

Fig. 5. Achelous spinimanus (Latreille, 1819). Seasonal variation in the proportion of adult females and adult males in different gonadal stages in the Ubatuba region (OF, ovigerous females; DE, developed gonads; ED, developing gonads; RU, rudimentary gonads; IM, immatures). 

Fig. 6. Achelous spinimanus (Latreille, 1819). Correspondence Analysis (CA) of the abundance of demographic groups in the seasons in 1998 and 1999 sampled in the Ubatuba region (J, juveniles; AM, adult males; AF, adult females; OF, ovigerous females). 

There was a significant correlation between three abiotic parameters (temperature, bottom salinity, and mean organic matter content) and the abundance of ovigerous females and juveniles (Tab. II). According to the RDA axis 1, which explained 75% of the variance, low bottom temperatures favored the abundance of ovigerous females, or at low temperatures there is an increased abundance of ovigerous females. The abundance of juveniles was associated with higher values of sediment organic matter contents and bottom temperature, sites with large amounts of organic matter and high temperatures probably have a higher number of juveniles.

DISCUSSION

Although ovigerous females of A. spinimanus were found at the 7.5 m deep sites, they were the most abundant demographic group in the deeper sites (15 and 20 m). This behavior which is consistent with the patterns proposed for crustaceans with planktotrophic larval stages. Because to the improvement of larval dispersion and survival, since environmental conditions are more stable at higher depths and thus the larvae suffer less physiological stress (^{Abelló, 1989}). Also, in deeper regions the larval dispersal is facilitated by oceanic currents and the predation pressure is less intense (^{Andrade et al., 2014}).

^{Bernardes et al. (2019}) suggested that ovigerous females of H. pudibundus are usually found in deeper sites because from the coast have higher salinity, which favors the embryo development during incubation. The differential occupation by different demographic groups has been previously recorded in other species. For instance, immature individuals of C. ornatus were more abundant in shallow waters near the coast (^{Andrade et al., 2014}). This habitat choice might be related to a higher food and shelter availability since shallow waters are more protected and have higher organic matter contents (^{Andrade et al., 2014}). These authors suggested that ovigerous females move away from the coast because their larvae would fluctuate more easily at higher salinities. A differential occupation pattern was also seen in a population of A. spinimanus in Fortaleza Bay, where immature individuals were more abundant in shallow areas (4 m of depth) (^{Santos et al., 1995}). The authors suggested that the higher productivity of shallow waters would lead to higher growth rates and favor the establishment of immature individuals. ^{Negreiros-Fransozo et al. (1991}), in a study in the Fortaleza Bay, evidenced that the sample area with the highest content of organic matter was the most appropriate place for the development and proliferation of organisms. Besides that, ^{Fransozo et al. (1992}) that organic matter content can modulate the occurrence and distribution of the Brachyura.

It is worth mentioning that the highest abundance of all demographic groups was recorded at 7.5 m, which is located within an area naturally excluded from fisheries. The absence of fishing might have contributed to this higher abundance of swimming crabs. Areas less impacted by fisheries have their habitat complexity preserved, which favors the establishment of new individuals (^{Kaiser et al., 2002}). According to ^{Fransozo et al. (2016}), the fishing activity is predatory and disturbs the benthic communities, since it lacks a defined target and mixes the sea bed, thus displacing or removing organisms from their habitats.

Our results indicate that the studied population has a continuous reproduction, since ovigerous females were found year-round. Extensive reproductive periods are characterized by several spawning events throughout the year (^{Giese, 1959}). A continuous reproduction is a common feature of tropical and subtropical marine brachyurans.

However, there are records of A. spinimanus reproducing seasonally, during summer and autumn, in Macaé, Rio de Janeiro (^{Andrade et al., 2017}). This record corroborates the data from the North Hemisphere where ovigerous females of this species were found only in the warmer months (^{Dudley & Judy, 1971}; ^{Camp et al., 1977}; ^{Olsen et al., 1978}). Thus, the reproductive pattern of A. spinimanus in Macaé (^{Andrade et al., 2017}) represents a physiologic adjustment to local conditions. According to the authors, although Macaé is less than 500 km distant from Ubatuba, it belongs to a resurgence area and has colder waters, similar to regions at higher latitudes where the reproduction is also seasonal (^{Andrade et al., 2017}). We can infer that the reproductive pattern of A. spinimanus is related to the latitude, since in the present study the individuals showed a continuous reproduction, however this may be variable depending on the location i.e., their metabolism responds differently depending on the region, affecting the reproductive characteristics. Similarly, ^{Pinheiro & Fransozo (2002}) and ^{Andrade et al. (2015b}) observed that an A. cribarius population adjusted to new environmental conditions and changed from a continuous to a seasonal reproduction to ensure a better nutrient supply to its larvae.

We found an association between abundance peaks of ovigerous females and low bottom temperatures, which were recorded in spring due to the SACW intrusion. The SACW has physical and chemical features that bring a nutrient enrichment to the coast, favoring the primary production (^{Castro-Filho et al., 1987}) and providing food supply to larvae (^{Vega-Pérez, 1993}). Therefore, SACW’s physico-chemical features are probably the real modulators of A. spinimanus reproductive period in our study region. Other authors also suggested that the number of ovigerous females increases in periods of higher primary productivity (^{Gonçalves et al., 2017}). According to these authors, this increase in periods of high phytoplankton levels is consistent with the patterns seen in crustaceans with planktotrophic larval stages. Since the phytoplankton form the base of marine food webs, serving as a food resource to several species with larval stages, their presence triggers several stimuli to the beginning of larval incubation (^{Starr et al., 1994}). However, it must be highlighted that the reproductive period of tropical species with continuous reproduction cannot be established based on one or few parameters, but on a complex group of variables. These variables may include physical and chemical parameters and interactions, such as competition, predation, and social structures.

Based on our results, A. spinimanus showed the continuous spawning pattern proposed by ^{Crocos & Van Der Velde (1995}). The continuous reproduction allowed the establishment of juveniles year-round, and the recruitment was higher in periods following higher spawning rates. Moreover, juveniles were more abundant in periods with high organic matter contents. Although swimming crabs pray on bivalves and gastropods, they also consume decomposed organic matter (^{Warner, 1977}).

We can infer that the dynamics of water masses and biological interactions within the benthic community are the possible modulators of the reproductive strategies of A. spinimanus. Some of the population features observed during our 2-year survey indicate that A. spinimanus uses the Ubatuba region to complete its life cycle. These features include: the continuous reproduction, the high abundance of immature individuals and ovigerous females, and the presence of individuals at all stages of gonadal development. Therefore, direct and indirect changes in the region must be monitored to avoid damaging the natural stocks of marine organisms, and especially of A. spinimanus, which is becoming a new target species. In addition, this study provides a basis for comparison with current data, and it also attests the efficiency of the implemented strategies in 2008-2009 for the species conservation.