INTRODUCTION
Most decapod crustaceans have separate sexes (gonochorism), but some caridean species exhibit sequential hermaphroditism in which there is sexual reversal at a certain life stage (Bauer, Reference Bauer2000). When the individual first develops as a male and then later changes to a female, the sexual system is termed protandry or protandric hermaphroditism (PH). For a small group of Caridea, an even more specific type of hermaphroditism is described, i.e. protandric simultaneous hermaphroditism (PSH, Bauer, Reference Bauer2000). In this sexual system, the individual develops and reproduces initially as a male but subsequently changes sex into a functional simultaneous hermaphrodite. These latter individuals (female phase or FP in Bauer & Holt, Reference Bauer and Holt1998; Bauer, Reference Bauer2000) have a primarily female phenotype, produce and incubate embryos, but they can also mate successfully as males. During sex change, male secondary sexual characters such as the appendix masculina, a typical feature of caridean males associated with spermatophore transfer (Bauer, Reference Bauer1976; Berg & Sandifer, Reference Berg and Sandifer1984), are reduced or lost. Female external characters associated with spawning and the incubation of embryos are developed, but the gonads are functional ovotestes (Bauer & Holt, Reference Bauer and Holt1998; Bauer, Reference Bauer2006; Baeza, Reference Baeza2009). This sexual pattern has been confirmed for all species of the genera Lysmata Risso, 1816 and Exhippolysmata Stebbing, 1915 and also for Parhippolyte misticia (Clark, 1989) (Laubenheimer & Rhyne, Reference Laubenheimer and Rhyne2008; Baeza, Reference Baeza2009; Braga et al., Reference Braga, López-Greco and Fransozo2009; Onaga et al., Reference Onaga, Fiedler and Baeza2012).
The spiny shrimp Exhippolysmata oplophoroides (Holthuis, 1948) is part of the by-catch taken in penaeid shrimp fisheries of high economic interest in Brazil (e.g. Xiphopenaeus kroyeri (Heller, 1862) and Litopenaeus schmitti (Burkenroad, 1936)) (Costa et al., Reference Costa, Fransozo, Mantelatto and Castro2000). Significant studies on E. oplophoroides include the Chacur & Negreiros-Fransozo (Reference Chacur and Negreiros-Fransozo1998) study on fecundity; the Negreiros-Fransozo et al. (Reference Negreiros-Fransozo, Gonzales-Gordilho and Fransozo2002) description of its first larval stage; the Fransozo et al. (Reference Fransozo, Costa, Bertini and Cobo2005) paper on population biology focusing on temporal distribution, size and reproductive period; Braga et al. (Reference Braga, López-Greco and Fransozo2009) and Laubenheimer & Rhyne (Reference Laubenheimer and Rhyne2008) demonstrated PSH in this species; and Baeza et al. (Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010) studied some aspects of population biology such as sex ratio age, mortality and reproductive period.
In this study, an E. oplophoroides population is studied for the first time near an upwelling area in which, despite its location at a tropical latitude (22°33′S), the shallow bottom water temperature does not exceed 21°C during most of the year (Silva et al., Reference Silva, Sancinetti, Fransozo, Azevedo and Costa2014; Pantaleão et al., Reference Pantaleão, Carvalho-Batista, Fransozo and Costa2016). In this area, the South Atlantic Central Water (SACW) promotes nutrient transport (N and P) from the bottom to the photic zone, directly influencing primary productivity (Odebrecht & Djurfeldt, Reference Odebrecht and Djurfeldt1996). This increased primary productivity can have a great influence on both zooplankton and benthic communities (Mann & Lazier, Reference Mann and Lazier1996). The concentration of chlorophyll-a in the water column may reach values 10 times higher than those in the Ubatuba region (De Léo & Pires-Vanin, Reference De Léo and Pires-Vanin2006), where all previous studies on E. oplophoroides have been performed.
The energy allocation for physiological processes such as growth and reproduction is an important aspect of an animal's life history (Schaffer, Reference Schaffer1983; Lika, Reference Lika2003). The allocation of resources for reproductive processes in the male or female is defined as sex allocation, and it may influence the population structure, affecting for example the sex ratio (Charnov, Reference Charnov1982). In PH and PSH species, the sex ratio can be biased toward the male or female (PH) or simultaneous hermaphrodite phase (PSH) according to resource availability (Charnov, Reference Charnov1982; Baeza, Reference Baeza2007a). The estimate of the size at which the sex change occurs and the conditions affecting this change are applications of sex allocation theory in the life history of an organism (Charnov, Reference Charnov1982; Hardy, Reference Hardy2002).
Considering the abiotic and oceanographic features of upwelling areas and the influence of these characteristics on populations, the present study aimed to analyse the population dynamics of E. oplophoroides, testing the hypothesis that upwelling has a significant effect, in comparison to non-upwelling areas, on reproductive period, sex ratio, growth rate, longevity, mortality, relative growth and the size at which the sex phase change occurs. Given the results of Zhang & Lin (Reference Zhang and Lin2004) on the role of pleopod appendices in copulatory abilities of PSH individuals, we also tested the hypothesis that growth of the appendices internae of the pleopods changed with sex change from the male to the simultaneous hermaphrodite phase.
MATERIALS AND METHODS
Sampling
Shrimps were collected monthly from July 2010 to June 2011 on the northern coast of Rio de Janeiro (Macaé – RJ, 22°33′S 41°78′W). Six locations were sampled: 3 at 5 m, 3 at 15 m. Sampling was done using a shrimp boat equipped with 10-metre-long double-rig shrimp trawls, with 20 mm net mesh and 18 mm cod-end mesh. Each location was trawled over a 30 min period at a constant speed of 2.0 knots, and ~18,500 m2 were covered in each trawl sample. After sampling, shrimps were bagged, stored in coolers with crushed ice, taken to the laboratory and later preserved in 70% ethanol, after measurements and observations were taken.
The carapace length (CL) of individuals was measured with a digital caliper to 0.01 mm. Individuals were classified as male or simultaneous hermaphrodite according to the developmental stage of the appendix masculina on the endopod of the second pleopod, i.e. well developed and with spines in the male phase and reduced and spineless in the hermaphrodite phase (Braga et al., Reference Braga, López-Greco and Fransozo2009).
Population dynamics
The population structure was assessed by the distribution of size frequency in different demographic categories (MP: male phase; HP: hermaphrodite phase without embryos; and HP-E hermaphrodite phase with embryos), using size classes with 1 mm intervals.
The reproductive period was calculated based on the frequency of the hermaphrodite phase with embryos in each month sampled. For the sex ratio, we used the Chi squared test (χ2) for goodness of fit (α = 0.05) (Sokal & Rohlf, Reference Sokal and Rohlf1995) to determine if the sex ratio was significant biased towards MPs or HPs throughout the study period.
Growth, longevity and mortality
Considering that E. oplophoroides is a protandric simultaneous hermaphrodite (PSH) species, the growth analysis was performed by grouping male and hermaphrodite phases. For each sample month, the length (CL) frequency was distributed in 1 mm size classes, and modes were calculated using the software ‘PeakFit’ (PeakFit v. 4.06 SPSS Inc. for Windows Copyright 1991–1999, AISN Software Inc.).
For estimates of growth parameters, all identified cohorts were adjusted to the growth model of Von Bertalanffy (Reference Von Bertalanffy1938): CLt = CL∞[1 − e −k (t−t0)], where the carapace length CLt is the estimated size at age t, CL∞ is the asymptotic size, k is the growth coefficient and t 0 is the theoretical point in time when the individual has zero length. Growth parameters were estimated for the different cohorts with the Excel tool ‘Solver’, varying the equation parameters CL∞, k and t 0. The selected cohorts were those consistent with the species' life history cycle, considering longevity, the growth coefficient k and asymptotic size (CL∞). Comparison of the growth curves was performed using an F test (P = 0.05) (Cerrato, Reference Cerrato1990). Longevity was estimated by the inverse equation of Von Bertalanffy, with modifications suggested by D'Incao & Fonseca (Reference D'Incao and Fonseca1999), considering t 0 = 0 and CL/CL∞ = 0.99. The longevity equation is given by: t = (t 0 − (1/k) Ln (1 − CLt/CL∞).
The empirical natural mortality (M) (Pauly, Reference Pauly1980) was calculated by the Beverton & Holt (Reference Beverton and Holt1959) method using the FISAT II program (Food and Agriculture Organization of the United Nations; http://www.fao.org/fishery/topic/16072/en#3).
Relative growth and size estimate of the sex change
For relative growth analyses, the following structures were measured in 407 individuals: AIL2-5 length of the appendix interna of the second to fifth pleopod; AML, length of appendix masculine; CL, carapace length; FPL, length of first pereopod; PL, length of second pleuron; SPL, length of second pleopod (Figure 1). The appendices internae link the endopods of each segmental pair of pleopods; the appendix masculina is a typical structure on the endopods of the second pleopods of caridean shrimp males (Bauer, Reference Bauer2004) which is thought to be involved in copulation and sperm transfer (Bauer, Reference Bauer, Thiel and Watling2013).
Fig. 1. Exhippolysmata oplophoroides (Holthuis, 1948). Body dimensions used in the morphometric analyses. (A) hermaphrodite carrying embryos, (B) magnification of appendix interna and masculina of the second pleopod, (C) second pleopod. AIL, length of appendix interna; AML, length of appendix masculine; CL, carapace length; FPL, length of first pereopod; PL, length of second pleuron; SPL, length of second pleopod. (Scale bar: A = 10 mm, B = 0.5 mm, C = 2 mm).
The relative growth analysis allows for detecting possible changes in the growth pattern of body structures as a function of an independent variable (CL). The allometric equation y = axb was used in the linearized version (log y = log a + b log x), in which y is the dependent variable (morphological structure), x is the independent variable (CL), b is the allometric coefficient and a is where the line intersects the y-axis. The allometric condition b for each structure was analysed (b = 1: isometry, b < 1: negative allometry, b > 1: positive allometry) with a Student's t-test (H o: b = 1; α = 0.05) (Zar, Reference Zar1996). The angular and linear coefficients a and b, respectively, from the male and hermaphrodite phases were subjected to an analysis of covariance (ANCOVA) test of homogeneity of slopes for each morphological structure in order to determine whether the MP and HP groups could be represented by separate linear equations.
To determine the size of sex change in the population, the transformed data (log10) were subjected to the non-hierarchical analysis ‘K-means clustering’. This method distributes data in groups of numbers previously established by an iterative process that minimizes variance inside groups and maximizes the variance among them. The classification result (K-means) was refined by applying a discriminant analysis. The size of the smallest individual classified by the discriminant analysis as belonging to the hermaphrodite phase was used as an estimated value at which the sexual change occurs. This statistical methodology was based on Sampedro et al. (Reference Sampedro, Gonzáles-Gurriarán, Freire and Muiño1999).
RESULTS
Population dynamics
A total of 2156 individuals were collected: 630 in the male phase (MP), 343 in the hermaphrodite phase without embryos (HP) and 1183 in the hermaphrodite phase with embryos (HP-E). The minimum and maximum sizes CL (mm) were 3.80 to 12.10 (7.98 ± 2.12) in the MP, 7.60 to 18.82 (9.97 ± 1.99) in the HP and 6.70 at 20:18 (11:49 ± 1.98) in the HP-E.
The highest frequency of individuals in the MP were observed in the 8–9 mm size classes (CL), while individuals in the HP had higher proportions in the 10–11 mm size classes (CL) (Figure 2). Two peaks in the MP were observed, one in September and another in May. Shrimp in the HP-E were present in all sample months and almost entirely with percentage values above 50%, indicating continuous reproduction throughout the year (Figure 3). There was a statistically significant difference in the sex ratio (P < 0.05) in favour of the hermaphrodite phase in almost all sample months, except for September 2010 (Table 1).
Fig. 2. Exhippolysmata oplophoroides (Holthuis, 1948). Size-frequency distribution of the male phase (MP) (black bars), hermaphrodite phase without embryos (HP) (white bars) and hermaphrodite phase with embryos (HP-E) (grey bars) (all individuals sampled: N = 2156).
Fig. 3. Exhippolysmata oplophoroides (Holthuis, 1948). Monthly per cent frequency of the male phase (MP) (black bars), hermaphrodite phase without embryos (HP) (white bars) and hermaphrodite phase with embryos (HP-E) (grey bars).
Table 1. Exhippolysmata oplophoroides (Holthuis, 1948).
Number of individuals in the male phase (MP) and number of individuals in the hermaphrodite phase (HP) from July 2010 to June 2011. The deviation from an equal sex ratio was tested for each month.
*Significant difference from MP/HP = 1.0.
Growth, longevity and mortality
Five cohorts were determined for the population of E. oplophoroides sampled in Macaé/RJ. The estimated growth parameters were k = 0.00576 mm day−1 (or k = 0.17 mm month−1), t 0 = −0.24, CC = 18.89 mm. The maximum longevity was estimated to be 2.19 years (Figure 4) and the natural mortality was 0.07 month−1 (M = 0.856 year−1).
Fig. 4. Exhippolysmata oplophoroides (Holthuis, 1948). Growth curves estimated for the population of Macaé, Rio de Janeiro state. The middle line is the mean and the outer lines are the 95% confidence limits.
Relative growth and estimated size of sex change
All relative growth equations for different morphological structures showed statistically significant differences between the male (MP) and hermaphrodite (HP) phases (ANCOVA P < 0.05) (Table 2). The relationship which best represents the sex change was AML vs CL, showing a reduction in the male appendix after change from the MP to the HP (Figure 5). The estimate for the size of sex change is 9.93 mm CL.
Fig. 5. Exhippolysmata oplophoroides (Holthuis, 1948). Estimated size at the change from the male to the hermaphrodite phase. The estimated size refers to the smallest individual after the inflection point of the phase equations for the male and hermaphrodite phases.
Table 2. Exhippolysmata oplophoroides (Holthuis, 1948).
Results of analysis of covariance (ANCOVA) tests for homogeneity of slopes to compare relative growth of possible secondary sexual characters between male and hermaphrodite phases.
AIL, length of appendix interna; AML, length of appendix masculine; CL, carapace length; FPL, length of first pereopod; PL, length of second pleuron; SPL, length of second pleopod; HP, simultaneous hermaphrodite phase; MP, Male phase.
Par = parameter, a = intercept, b = slope, *P < 0.05.
The PL vs CL relationships indicated positive allometric growth in both phases. Growth was negatively allometric for the relationships FPL vs CL and AML vs CL. For the relationships SPL vs CL, AIL2 vs CL, AIL3 and AIL5 vs CL, negative allometric growth in the male phase was followed by positive allometric growth in the hermaphrodite phase. For the relationship AIL4 vs CL, negative allometric growth was observed in the male phase followed by isometric growth. A detailed description of each relationship can be found in Table 3.
Table 3. Exhippolysmata oplophoroides (Holthuis, 1948).
Regression analysis of morphometric data.
AIL, length of appendix interna; AML, length of appendix masculine; CL, carapace length; FPL, length of first pereopod; PL, length of second pleuron; SPL, length of second pleopod; HP, simultaneous hermaphrodite phase; MP, Male phase, + = positive allometry, 0 = isometry, − = negative allometry.
DISCUSSION
The size distribution of the male phase (MP) and simultaneous hermaphroditic phase (HP) in E. oplophoroides, with a sex change from smaller MPs to larger HPs, is concordant with the size advantage model described by Ghiselin (Reference Ghiselin1969) and Warner (Reference Warner1975), as well as sex allocation models (Baeza, Reference Baeza2007a). For Lysmata wurdemanni (Gibbes, 1850), another species with protandric simultaneous hermaphroditism (PSH), it was found that even the smallest male-phase individuals are able to copulate with larger hermaphrodites. The adaptive advantage of small male size in carideans is that they may be more cryptic and thus less vulnerable to predators, and have lower energy needs. These are advantages if males do not defend territory or compete agonistically for females, i.e. a ‘pure search’ (promiscuous) mating system (Wickler & Seibt, Reference Wickler and Seibt1981; Bauer, Reference Bauer2004). Lysmata wurdemanni (Bauer & Holt, Reference Bauer and Holt1998; Bauer, Reference Bauer2006) and presumably E. oplophoroides (Laubenheimer & Rhyne, Reference Laubenheimer and Rhyne2008) have such a mating system. Female fecundity of the HPs in L. wurdemanni increases with increasing size (Bauer, Reference Bauer2005), as in other caridean shrimps (Bauer, Reference Bauer2004). We assume a similar relationship of size and sex allocation in E. oplophoroides.
A continuous reproductive period was observed, with hermaphrodite shrimp carrying embryos in all months sampled. In carideans with this type of reproduction, ovarian development usually occurs while embryos are incubating (Bauer, Reference Bauer2004). Thus, soon after larval hatching, hermaphrodites of E. oplophoroides can copulate and produce a new brood of embryos. The ability of individuals in the hermaphrodite phase to reproduce as males or as females may also favour continuous reproduction.
Although the area of the present study has cooler waters than expected at the latitude studied due to upwelling (Silva et al., Reference Silva, Sancinetti, Fransozo, Azevedo and Costa2014; Pantaleão et al., Reference Pantaleão, Carvalho-Batista, Fransozo and Costa2016), the monthly frequency of hermaphrodites with embryos was usually above 50%, similar to that found by Baeza et al. (Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010) for the same species in the Ubatuba/SP region. Considering that this is a PSH species, in which smaller individuals (new recruits) are in the male phase, we can infer from our data that recruitment was also continuous, but with a peak in September.
The sex ratio was biased towards hermaphrodite phase in the present study, as found by Baeza et al. (Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010) for E. oplophoroides in the Ubatuba/SP region. In the absence of mating opportunities, male-phase shrimps can accelerate their development and change to simultaneous hermaphrodites more quickly (Baeza & Bauer, Reference Baeza and Bauer2004), which may explain the dominance of the hermaphrodite phase. Thus, individuals increase their reproductive potential and can act as males or as females, according to the population structure and whether conditions favour behaving as a male or a female.
However, in a study focusing on sex allocation in L. wurdemanni, initial males delayed their development when there was an increase in hermaphrodites in the population, which can reflect a response and/or phenotypic flexibility to environmental conditions (Baeza, Reference Baeza2007b). The results discussed above concerning the sex ratio reinforce the phenotypic plasticity of PSH species and their ability to change the sex ratio according to resource availability in the environment.
Concerning the maximum body size, k and longevity of E. oplophoroides compared with that reported by Baeza et al. (Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010), the greatest difference was with regard to age and lifespan. Longevity showed considerable differences (~2 years) in two closely adjacent bays in the Ubatuba region (Table 4). This large difference in longevity in the Ubatuba population might be explained by variations in sediment characteristics and organic content, which could affect food availability and the presence or absence of predators. The values of k and mortality might explain such differences, even though the techniques employed for such calculations were different in our study from that used in Baeza et al. (Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010). There is a negative correlation between k values and longevity. The physical features of the Macaé region, such as lower temperatures and higher primary productivity when compared with the Ubatuba region (De Léo & Pires-Vanin, Reference De Léo and Pires-Vanin2006), are probably the main factors that influenced larger sizes and constant growth. The natural mortality was also lower. In contrast, the mortality rate in Mar Virado Bay was very high (Table 4), although the k and longevity values were similar in both regions.
Table 4. Growth parameters of Exhippolysmata oplophoroides (Holthuis, 1948) from Macaé/RJ (present study), Mar Virado and Ubatuba, on the north-eastern coast of São Paulo State, Brazil (Baeza et al., Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010).
As with the growth analyses, the estimated size at which the change from the male to the simultaneous hermaphrodite phase occurs showed higher values when compared with those found by Baeza et al. (Reference Baeza, Braga, López-greco, Perez, Negreiros-Fransozo and Fransozo2010) in Ubatuba. Possible physiological adaptations to different environmental conditions could explain these differences in maximum size, growth rate, mortality and size at the sex change in both regions. Different conditions of temperature and photoperiod had an effect on the sex change of L. wurdemanni under laboratory experiments (Bauer, Reference Bauer2002; Baldwin & Bauer, Reference Baldwin and Bauer2003). Besides environmental conditions, social interactions in the population also may have a great influence on the size of sex change (Charnov et al., Reference Charnov, Gotshall and Robinson1978; Baeza & Bauer, Reference Baeza and Bauer2004).
Both the male and hermaphrodite phases exhibited positive allometric growth in the length of second abdominal pleura. In caridean females, increase in the relative size of the first three abdominal pleura is an adaptation for brooding embryos, so an increase in the growth of this structure is one of the factors related to reproductive success in the hermaphrodite phase (Bauer, Reference Bauer2004). Thus, morphological preparation for the incubation of embryos during the female (simultaneous hermaphrodite) phase begins during the male phase.
Negative allometric growth was found in relation to the first pereopod length in the male and hermaphrodite phases. However, the second pereopod showed negative allometric growth in the male phase followed by positive allometric growth in the hermaphrodite phase. The first two pereopods in carideans are chelate; they are fundamental in activities such as food searching and handling, defence, territorial disputes and grooming (Bauer, Reference Bauer2004). Grooming behaviour reduces debris fouling on the body surface and thus prevents sensory and locomotion disabilities (Bauer, Reference Bauer1978). The positive allometry observed in the second pereopod, the grooming appendage in hippolytid carideans, is also an advantage in embryo incubation. In females with embryos, the chelae of the second pereopods are used for cleaning the mass of embryos, removing sediment and unfertilized eggs and thereby preventing bacterial growth in fertilized eggs (Bauer, Reference Bauer2004). An increase in the size of this structure in the hermaphrodite phase, besides being an advantage in the care of embryos, might also improve food collection, especially in a phase with high energy demand for embryo production.
The hermaphrodite phase showed a reduction in the appendix masculina with increased body size. In simultaneous hermaphrodite shrimps, a change from the male to the hermaphrodite phase is accompanied by changes in some structures, including a reduction in the size of this male appendix and the number of its spines (Bauer & Holt, Reference Bauer and Holt1998; Bauer & Newman, Reference Bauer and Newman2004). Previous studies on caridean shrimps have indicated that the appendix masculina is important in transferring spermatophores from the male to the female during copulation (Bauer, Reference Bauer1976; Berg & Sandifer, Reference Berg and Sandifer1984). However, Zhang & Lin (Reference Zhang and Lin2004) found that the second pleopods (with appendices masculinae) are not necessary for successful copulation in either phase in L. wurdemanni. Thus, the appendix may be a vestigial structure in the hermaphrodite phase without selective pressure to maintain it (Bauer, Reference Bauer2000). However, this does not explain its presence in the male phase, given the experiments of Zhang & Lin.
The appendices internae of the second to fifth pleopods exhibited changes in the growth pattern (allometry) between the male and hermaphrodite phases. This pattern did not support the hypothesis that an enlarged appendices internae might be a substitute for the appendices masculinae, which are reduced or lost in the hermaphrodite phase. Pleopods of a segmental pair are linked by the cincinnuli of the appendices internae and help in the synchronized movement of pleopods during swimming (Bauer, Reference Bauer1976). One hypothesis for the positive allometry in this structure in the hermaphrodite phase is that it may provide an increased volume of the incubation chamber for embryos among pleopods, thus increasing the reproductive capacity.
Local environmental features such as lower temperatures and high primary productivity due to the Cabo Frio upwelling influence resulted in larger sizes, slower growth rates and lower mortality for E. oplophoroides when compared with this species in the Ubatuba/SP region. The high primary productivity in the study area may have also contributed to a sex ratio biased towards HPs, since it provides the increased food and energy needed to sustain embryo production. Thus, the environmental conditions associated with upwelling resulted in reproduction and growth more characteristic of higher latitudes.
ACKNOWLEDGEMENTS
Thanks are due to all members of the LABCAM, and to Dr Alexandre Azevedo and Dr Gustavo Sérgio Sancinetti for their help during fieldwork. We also thank the Universidade Federal do Rio de Janeiro/NUPEM for providing the infrastructure to carry out this work at Macaé, and the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA) for granting us permission to collect the shrimp (SISBIO N°. 23012-1). This is Contribution No. 185 of the Laboratory of Crustacean Research (R.T. Bauer), University of Louisiana, Lafayette.
FINANCIAL SUPPORT
The authors are grateful for funding from the São Paulo Research Foundation – FAPESP (AR #2009/54672-4 and Biota Temático 2010/50188-8 to RCC, and a scholarship #2013/12136-4 to RAP, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico – CNPq (Proc. # 406006/2012-1 and Scholarship 1D # 305919/2014-8 to RCC and Scholarship #130837/2011-3 to JAFP).
