Color variation of the freshwater crab Dilocarcinus pagei Stimpson, 1861 captured in the Amazon region at Itacoatiara, Amazonas, Brazil

The relationship between color and size supports the hypothesis that color is an important signal for some crab species during mating rituals or agonistic interactions. Thus, the objective of this study was to describe the color variation of D. pagei and its relationship with sex and sexual maturity. The crabs were placed on a surface for photographic recording of the body parts: cephalothorax, chelipeds, abdomen and pereiopods using a digital camera installed on a photographic table. Images were converted to the spectrum of red, green, blue and also of them combined (RGB). The values of each spectrum revealed that the color variation of the cephalothorax and chelipeds were different between adults and juveniles. The abdomen and pereiopods showed color variation in both sexes and at developmental stages. The pattern of color variation observed in juveniles ranged from light to dark brown and adults are from orange to intense red. The change in color of the cephalothorax and chelipeds could be used as a character to determine the onset of sexual maturity. The high color variation of the abdomen and pereiopods in both sexes and at developmental stages, though, indicates individual variation which probably is associated with other factors, such as molt stage or even the quality of food available.

According to Detto et al. (2004), the existence of a relationship between color and individual crab size supports the hypothesis that color is an important signal for the crab Heloecius cordiformis (H. Milne Edwards, 1837) during mating rituals or aggressive interactions. In Austruca mjoebergi (Rathbun, 1924), females use male cheliped color, regardless of their specific waving display, to identify them as members of the same species (Detto, 2007). Males of Tubuca capricornis (Crane, 1975) recognize neighboring females based on the distinct color patterns of their cephalothorax (Detto et al., 2006).
Dilocarcinus pagei is one of the most studied freshwater crab species in Brazil, and there is already published information on population dynamics , behavior (Affonso and Signorelli, 2011;Sant'Anna et al., 2014;Senkman et al., 2015), experimental culture (Sant'Anna et al., 2015), and sexual maturity and dimorphism, (Davanso et al., 2016), but there is no information on the coloration patterns of this species. Thus, this study evaluated the color variation in D. pagei and its relationship with the sex and sexual maturity of these crabs.

Collection of samples
The specimens were collected in the municipality of Itacoatiara (AM) in a floodplain area called Poranga (03°07'11.4" S; 58°27'1.0" W), between August 2016 and April 2017 (Fig. 1). The crabs were collected on the sediment surface and among the adjacent macrophytic vegetation. The crustaceans were packed in plastic boxes with water and aeration and transported to the laboratory.
In the laboratory, the animals were identified according to the morphological characteristics described by Magalhães (2003). The crabs were sexed by the inspection of abdomen morphology and all animals was measured with a caliper (0.05 mm). The size of the carapace width was used to separate the onset of morphological sexual maturity, which was previously determined to be 28.7 mm for males and 24.2 mm for females (Davanso et al., 2016).

Image and color analysis
The crabs were photographed using a NIKON Coolpix L810 digital camera with 4.0-104 mm lens, attached to a specific table with a fixed height of 20 cm from the base, and with lighting from two 20 W fluorescent lamps, behind a refractory surface to avoid reflections.
The camera was adjusted to capture standard white balance images without additional color filtering and with a shutter speed of 1/1500 s to standardize the image brightness error. Images were converted to red (R), green (G), blue (B) and combined RGB values (interaction of the values of the three colors).
The recorded values of the images were collected from a histogram provided by free software GIMP (GNU Image Manipulation Program, EUA) obtained from the selected area of each morphological structure (cephalothorax surface, external surface of chelipeds, abdomen and pereiopods). This software has the same accuracy as other software described by Watanabe et al. (2015) who investigated the coloring pattern of the crab C. hellerii.
Digital images were individually analyzed using the color spectra of red, green and blue values ranging from 0 to 255. The RGB system combines the color spectra (Red, Green and Blue) in values of intensity with ranges from 0 to 255 (8 bits) for each color. The combination of values represents the individual colors with high values representing light colors and low values corresponding to dark colors (Casariego et al., 2011;Nasir and Faulkes, 2011).

Statistical analysis
The data for each color spectrum (R, G, B and RGB) from the images of the morphological structures were submitted to the Kolmogorov-Smirnov normality test. As the values did not show a normal distribution (P<0.05), a transformation by square root was applied to increase the normality of the distribution.
The influence of sex and developmental stage on color variation of D. pagei was verified by the mean value of each color spectrum (R, G, B and RGB). Each morphological structure (cephalothorax, abdomen, external face of chelipeds and pereiopods) were

Color variation of the crab Dilocarcinus pagei
Nauplius, 28: e2020026 submitted to analysis of variance (ANOVA two-way) considering sex (male and female) and developmental stage based on the size of the onset sexual maturity (juveniles and adults).

resulTs
A total of 186 crabs were collected (100 males and 86 females) and the mean size registered for each sex and developmental stage was similar (Tab. 1). The visible color variation of Dilocarcinus pagei shows two morphotypes at each developmental stage, with juveniles ranging from light to dark brown, and adults separated into the groups light orange to red (Fig. 2).
The color variation pattern observed in the developmental stage indicated a significant difference between juveniles and adults. The cephalothorax and chelipeds show differences between the adults and juveniles, but with no differences between sexes (Tab. 2). In the same table, the color of the abdomen and pereiopods was different between the sexes and in sexual maturity.
The means of the combined RGB spectra from the cephalothorax and chelipeds were higher in adults than in juveniles, indicating a darker color in adults and a lighter color in juveniles (Tab. 3). This same pattern of color spectrum was observed in the green and blue spectra. The red spectrum, though, was similar between the developmental stages.
The abdomen shows different colors in adult males with lower values of combined RGB spectra when compared to juvenile males and all females (adults and juveniles). The red color of the abdomen in adult males was confirmed by the lower values of the green and blue spectra (Tab. 4).
The values of the combined RGB spectra from the pereiopods demonstrated a different color between adults and juveniles, with adults being darker than juveniles. The same pattern was seen for the individual red, green and blue spectra (Tab. 4).

disCussion
The external color of D. pagei varies from light brown to red, confirmed by the different values in the green and blue color spectra. This color variation is associated with growth of morphological structures. According to Rao (2001), the movement of pigments promotes a dispersion or concentration at the epithelial chromatophores causing the color changes in crustaceans.
The color variation between adults and juveniles of D. pagei is related to pigment change after reaching sexual maturity, with adults showing dark colors and juveniles are lighter in color. In addition, color intensity may also vary during molting with adjustments in chromatophores, as seen by Casariego et al. (2011), with color associated with the interval of molting period.
In this study, adults of D. pagei showed post-moult colors of orange, brown and red. In Carcinus maenas (Linnaeus, 1758) color change was found in females during periods of high molting frequency after mating; becoming red in the intermolt periods and green after molting (Lee and Vespoli, 2015). The color of the cephalothorax in Cardisoma guanhumi Latreille 1825 could be associated with developmental stage because there is a relationship with molting frequency and the size at onset of sexual maturity (Silva et al., 2014). Adults of D. pagei have cephalothorax shades of red and orange, suggesting that these differences may influence the behavior of individuals as shown by Casariego et al. (2011) and Lee and Vespoli (2015) who all reported the importance of cephalothorax color variation in crab social interactions.
Males and females of D. pagei have different color variations on their abdomen, with females showing a red background with white spots, while males are fully intense red. This information could be important to help identify the onset of sexual maturity, since the color of the juvenile abdomen is completely brown in both sexes.
The mangrove crab Austruca perplexa (H. Milne Edwards, 1852) has the abdomen color as an important indicator of sexual maturity or sex, because it relies more on visual than chemical cues (Takeda, 2006). According to the same author, the intense color of adult females is a character used to distinguish mature from immature females, as well as assisting in visual identification of other sympatric crab species. The presence of white spots on the abdomen of D. pagei females may be caused by natural wear during locomotion. The abdomen experiences positive allometric growth because it has the function of incubation chamber during reproduction (Davanso et al., 2016). This positive allometric growth may alter the concentration of chromophores in the abdomen as observed in C. hellerii and A. cribrarius by Watanabe et al. (2015) and Pinheiro and Taddei (2000), respectively. The color variation of the chelipeds and pereiopods of D. pagei shows the same pattern as the cephalothorax. Juveniles present a brown coloration without difference between sexes, while adults present colors ranging from red to orange. This pattern is associated with the diverse functions of chelipeds during development, such as in females where they may promote protection of eggs and juveniles stored on the abdomen against predators (Daniels, 2001), and in males where they are used in mating behavioral displays (Herrera et al., 2013).
The color variation during ontogeny observed in D. pagei may be associated with the growth of these morphological structures, as shown in studies with the crab H. cordiformis, whose color is related to growth and may be promoted by foraging habits or the acquisition of sexual maturity (Detto et al., 2004).
The positive and negative relationships of color intensity recorded in D. pagei may further indicate the existence of a hierarchy in this species and could be an important character during territorial disputes or choice of partners; as noted in the blue crab Callinectes sapidus Rathbun, 1896 (Baldwin and Johnsen, 2012). Watanabe et al. (2015) suggests that the color variation related to the size of C. hellerii may indicate a dominance hierarchy among individuals for resources such as food and females available for mating (males only).
Dilocarcinus pagei turns progressively red with increasing size, indicating an association with the onset of sexual maturity and this can be used as a character to identify an adult or those close to adulthood. The red color in larger D. pagei individuals may have the function of facilitating the choice of partners by visual stimulation during the mating period, as well as promoting greater social interaction. This red color has as a disadvantage though, in the perceived increased risk of predation when such brightly colored crabs become easier to identify in vegetation and on substrate by predators. Other brachyurans also change their colors under predation risk through the modification of growth rates caused by stress (Henmi et al., 2006). Silbiger and Munguia (2008) reported that seasonal temperature changes can affect growth and promote color variation in mangrove crabs. However, further studies are needed to better understand how environmental parameters may influence Dilocarcinus pagei color variation.