Population dynamics of the pea crab Austinixa aidae ( Brachyura , Pinnotheridae ) : a symbiotic of the ghost shrimp Callichirus major ( Thalassinidea , Callianassidae ) from the southwestern Atlantic

The Pinnotheridae family is one of the most diverse and complex groups of brachyuran crabs, many of them symbionts of a wide variety of invertebrates. The present study describes the population dynamics of the pea crab Austinixa aidae (Righi, 1967), a symbiont associated with the burrows of the ghost shrimp Callichirus major (Say, 1818). Individuals (n = 588) were collected bimonthly from May, 2005 to September, 2006 along a sandy beach in the southwestern Atlantic, state of São Paulo, Brazil. Our data indicated that the population demography of A. aidae was characterized by a bimodal size-frequency distribution (between 2.0 and 4.0 mm and between 8.0 and 9.0 mm CW) that remained similar throughout the study period. Sex ratio does not differ significantly from 1:1 (p > 0.05), which confirms the pattern observed in other symbiontic pinnotherids. Density values (1.72 ± 1.34 ind. • ap. -1 ) are in agreement with those found for other species of the genus. The mean symbiosis incidence (75.6%) was one of the highest among species of the Pinnotheridae family, but it was the lowest among the three studied species of the genus. Recruitment pattern was annual, beginning in May and peaking in July, in both years, after the peak of ovigerous females in the population (from March to May). Our findings describe ecological and biological aspects of A. aidae similar to those of other species of this genus, even from different geographic localities.

Pinnotheridae pea crabs (Brachyura) are typically small and symbiotic of a wide variety of benthonic invertebrate hosts (SCHMITT et al., 1973;WILLIAMS, 1984;HARRISON & HANLEY, 2005).They are endo and/or ectosymbionts found associated with bivalves, gastropods, echinoderms, echiurids, brachiopods, balanoglossids and ascidians, and inside polychaets' tubes and crustacean burrows (SCHMITT et al., 1973;MANNING & MORTON, 1987;CAMPOS, 1990;HARRISON & HANLEY, 2005).Moreover, pinnotherid crabs are widely distributed along the marine coastal habitats of almost all oceans (SCHMITT et al., 1973;MARTINS & D'INCAO, 1996).Despite their ecological importance and their great diversity, the information about the population demography of the pea crabs is still scarce, especially about those species that inhabit the Atlantic coast of the Americas.
Austinixa Heard & Manning, 1997 species are found on sandy beaches and own peculiar ecological features, such as the symbiotic life with thalassinoid crustaceans of the Callichirus major (Say, 1818) complex or with few ecologically equivalent species (HEARD & MANNING, 1997;HARRISON, 2004), where they inhabit the upper portion of the host's burrows (MANNING & FELDER, 1989) and shelter near the entrance of these structures (Douglas F. Peiró, J. Antonio Baeza & Fernando L. Mantelatto, unpublished data).Studies that investigate the ecology of the sandy beaches indicate that the pinnotherid crabs from the Austinixa genus (= Pinnixa) are the frequent dominant infauna of the Americas (MANNING & FELDER, 1989;SOUZA & GIANUCA, 1995).The body feature of Austinixa (much wider lengthwise and with the third pair of walking legs more developed) is an adaptation to their symbiotic life style (HEARD & MANNING, 1997).All morphological adaptations facilitate their typical lateral locomotion into the confine narrow pipes and galleries of their hosts (HEARD & MANNING, 1997).Some of these features, shared with Pinnixa White, 1846, distinguish them from other brachyuran crabs (MCDERMOTT, 2006).
Austinixa aidae (Righi, 1967) has been reported as a symbiont of C. major, a callianassid shrimp that inhabits the intertidal zone of many beaches in Brazil (RODRIGUES & SHIMIZU, 1997).This species owns an Occidental Atlantic geographic distribution, from the state of Amapá to the state of Rio Grande do Sul (Brazil) (MELO, 1996;COELHO, 1997).Recently, its northern edge of distribution has been expanded until Tobago (Caribbean Sea), due to a synonymy with A. hardyi Heard & Manning, 1997, as described by HARRISON & HANLEY (2005), who found neither morphological nor molecular significant differences to support the existence of the two species.NG et al. (2008) also considered the synonymy of this species, but PALACIOS-THEIL et al. (2009) did not agree with HARRISON & HANLEY (2005) about the synonymy of A. aidae with A. hardyi.
Based on this promising scenario for investigation, this study describes the population dynamics of the symbiotic crab A. aidae in southeastern Brazil.The specific goals were to know more about size frequency distribution, sex ratio, density and mean incidence (percentage of occupation) in its hosts' galleries.The relation between density and size of the host, and reproductive and recruitment periods were also determined.Considering the morphological and ecological (symbiotic) intra-generic similarity in Austinixa, we evaluated these similarities' hypothesis in the population and in the biology of A. aidae and compared them to other species of this group using other available studies of the scientific literature -H 0 : there are differences among the species; H 1 : there are no differences among the species.

MATERIAL AND METHODS
Samples and procedures.The Perequê-açu beach (Ubatuba, northern coast state of São Paulo, Brazil; southwestern Atlantic), is located in the northern extremity of Ubatuba Bay.This beach is semi-protected and dissipative, composed by fine sand.Sampling was conducted bimonthly in the intertidal zone of the beach at daytime and during low tide, from May, 2005 to September, 2006.It was taken from a plot that was ~ 400 m long by ~ 30 m wide, parallel to the waterline, by the right side of river Indaiá's mouth (23°24'59''S, 45°03'17''W).
The crustaceans were collected for 2 hours during each sampling visit by two people with a commercial yabby pump (MANNING, 1975), used for sampling the C. major galleries.Once collected, the specimens were carefully washed with seawater and separated from the sand with a 1 mm mesh sieve and were directly placed into plastic bags, then labeled, frozen, and transported to the laboratory to be analyzed.Preliminary attempts permitted us to perfect the protocol above for shrimp and crab collections.We are confident that we were very efficient in capturing all animals from each gallery (D.F. Peiró, J. A. Baeza & F. L. Mantelatto, unpublished data).All material was preserved in ethanol 80% and deposited in the crustacean collection of the Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, (CCDB/ FFCLRP/USP) under the catalogue # 2102.In each sampling period, water temperature and salinity were measured before the collection.
The burrows of C. major own only one aperture to the sand surface that varies in shape and has a ~ 5 mm diameter.Behind their opening, the burrows resemble a narrow tube that runs perpendicular to the surface up to 20 or 40 cm deep.Austinixa aidae inhabits this tubular section of the burrow and not the sections below it (D.F. Peiró, J. A. Baeza & F. L. Mantelatto, unpublished data).From this point below, the diameter of the gallery increases; it directs to the sides and deepens farther.The length and the diameter of this last section are proportional to the size of the shrimp that constructs the gallery and it attains approximately 1.5 m depth and between 10 and 30 mm diameter (RODRIGUES & SHIMIZU, 1997).
During sampling, each burrow opening was pumped only twice, since the sediment collapses due to the fine grains of the sand.However, the rapid collapsing of the sediment during the process of pumping does not affect the ability to catch the crabs, given that these crabs only inhabit the narrower upper part of the pumped burrows.Importantly, the rapid collapse also eliminates the risk of sampling the same burrow more than once (ALVES & PEZZUTO, 1998a).Taking into consideration that the galleries of C. major have a single opening to the sand surface (SHIMIZU & ROSSO, 2000), the values reported herein refer always to A. aidae per gallery of C. major (ind.•ap. - ) (ALVES & PEZZUTO, 1998a).
Three ontogenetic stages were considered: recruits, juveniles and adults.The recruits -defined as those individuals that survived the pelagicbenthic transition and became first crab instars -correspond to the smallest individuals collected, which formed the first size class in the size structure.In essence, recruits are the early juveniles or the young of the year (PARDO et al., 2007).Juveniles were all non identified individuals that displayed undifferentiated or not well-developed pleopods, including the recruits.Adults were classified as either males or females according to the analysis of the pleopod.The males have a pair of long and thin modified pleopods on the ventral surface of the first abdominal somite and a pair of short modified pleopods on the second abdominal somite.The females have four pairs of short pleopods from the second to the fifth abdominal somites.The ovigerous females carry an egg mass adhered to the pleopods.The following measurement was performed under a stereomicroscope with the aid of a drawing tube (0.1 mm precision): the maximum carapace width (CW) of all crabs retrieved from each gallery was measured.The oval area length (OL) of the Thalassinidea hosts was measured with a 0.02 mm precision caliper.
Data analysis.In order to evaluate the correlation between the environmental factors (water temperature and salinity) and the abundance of crabs, the Pearson's product moment correlation was utilized.The Normality Test KS (Kolmogorov-Smirnov) was used to evaluate the normality of the population according to the size classes' structure.The number of classes was determined by the STURGES (1926) formula: k = 1 + Log 2 n; "k" is the number of classes and "n" is the size of sample.
The temporal patterns in the mean densities (number of A. aidae per gallery) and the incidence (percent of galleries occupied by A. aidae) were analyzed.Possible relations between densities and mean sizes of crabs and the size of their hosts were checked by the Pearson's product moment correlation (ALVES & PEZZUTO, 1998a).Differences from the expected 1:1 sex ratio pattern were checked for the total of samples collected and for each sampled month by the χ 2 text (WENNER, 1972).
Reproductive periods and recruitment were identified by the proportion of ovigerous females in relation to the total of females (PINHEIRO & FRANSOZO, 2002) and to the presence of juveniles in the samples (ALVES & PEZZUTO, 1998a), respectively.All statistical tests were conducted according to ZAR (1996), adopting the significance level p < 0.05.
The maximum number of individuals collected per gallery was 14 crabs.The total mean density was 1.72 ± 1.34 ind.• ap. - .The lowest mean density was registered in September, 2006 (1.3 ± 0.5 ind.• ap. - ) and the highest in July, 2006 (2.52 ± 1.94 ind.• ap. - ), with a particularly clear juveniles' variation (Fig. 2) during the sample period.The total mean incidence -percentage of occupation -of crabs in the galleries was 75.6%, with a variation during the sample period (Tab.I; Fig. 3).A significant positive correlation was found between the density (ind.• ap. - ) of A. aidae and the size (OL) of their respective hosts C. major (r = 0.501; p < 0.05; n = 73), i.e. the larger the hosts, the higher the number of symbiotic crabs in their burrows.On the other hand, no correlation was found between the size of crabs and the size of their respective hosts (OL) (r = 0.037; p > 0.05).The highest incidence of juveniles followed the months with a higher density of ovigerous females in the population (Figs 2, 3).It started in May and reached a higher proportion in July, in both years.Males and females did not present great peaks of density, the males' ranging from 0.50 to 1.13 ind.• ap. - (0.73 ± 0.20 ind.• ap. - ), and the females' from 0.58 to 0.97 ind.• ap. - (0.77 ± 0. 14 ind.• ap. - ).Regarding the males, the tendency of density increase occurred in November, 2005, four months after the recruitment of juveniles; and the second in July, 2006, subsequent bimonthly to the coming of the juveniles to the population.Regarding the females, four light tendencies of density increase (July andNovember, 2005, andMarch andJuly, 2006) occurred.Males and females presented a higher mean of incidence (54.5 ± 18.4% and 57.7 ± 14.9%, respectively) than the juveniles (17.8 ± 20.2 %); however the percentages of occupation vary correspondently during the sample period.The values of juvenile density fluctuated from 0 to 0.64 ind.• ap. - (0.23 ± 0.24 ind.• ap. - ).
Reproductive period and recruitment.The percentage of ovigerous females in relation to the total of females was 20.8%; the frequency varies from 0 (July, 2005) to 50% (May, 2005) among the samples (Tab.I; Fig. 5).The ovigerous females occurred in almost all sample months, except in July, 2005.Seasonal increases in frequency and abundance of this group were identified, with expressive peaks in 2005 (May and November: 50.0 and 41.2%, respectively), and in 2006 (between March and May: 31.6 and 29.6%, respectively).Due to the presence of ovigerous females throughout the year and the seasonal peaks, the reproductive period was characterized as seasonal continuous.This reproduction system can supply a constant larval input reflecting in continuous recruitment, but not observed in this study.It is important to point out in our study that the recruits were identified only in the months subsequent to the peaks of ovigerous females.The recruitment starts in May and attains major proportion in July, in both years, and it contributes to increase the abundance in the subsequent size classes in the subsequent sample months (Figs 6, 7).The juveniles were present in almost all samples, except in March, 2006, with seasonal abundance increases.The peaks of juveniles occur in May and July, in both years, in the months subsequent to the ovigerous females' peaks (Fig. 8) (remembering that the recruits are included in this classification).An exception occurred thereafter November, 2005 ovigerous female peak (the month with higher incidence), but subsequent months there are not a peak of juveniles.7) corroborated the same pattern identified by density (Fig. 2) and incidence (Fig. 3) analyses, with the seasonal continuous reproduction and the input of juveniles occurring throughout the year.Two courts were also identified in some sampled periods (Figs 6,7).

DISCUSSION
Population structure.It was detected that the dynamics of Austinixa aidae followed the pattern observed for congeneric species inhabiting different regions and different hosts, with some peculiarities.This profile can be hypothesized as adaptations for living in a different host and with polygynandrous mating systems (D.F. Peiró, J. A. Baeza & F. L. Mantelatto, unpublished data).The population of A. aidae shows common symbiotic characteristics that had been observed in a few other pinnotherid of the genus.At Perequê-açu beach, these crabs did not show correlation with the environmental factors.Nor did the ovigerous females separately, which can be more subject to the action of these factors (SASTRY, 1983).The absence of correlation may be related to the water temperature and salinity measurement method, which was performed only before the collections.This method cannot reflect the values that represent the sampled months.Moreover, tropical and subtropical regions present minor seasonal variation Population dynamics of the pea crab Austinixa aidae (Brachyura... in environmental factors, with fall variations during the year and, in some cases, no correlation can be detected (AYRES-PERES & MANTELATTO, 2008;MIRANDA & MANTELATTO, 2009).
A bimodal distribution frequency of individual size in the A. aidae population -with two peaks in the size class structure -reflects two reproductive and settlement pulses per year, one of them more intensive, accumulating individuals in higher size classes (where the growth stops), besides the differentiated growth between males and females.The continuous presence of individuals in all size classes in the bimonthly size-class frequency distribution (except in September, 2005 and2006) is a strong argument against the hypothesis of catastrophic mortality (DÍAZ & CONDE, 1989).Considering that the present study has provided no information about behavioral and mortality differences between sexes, the bimodal condition could be explained by the intensive settlement in certain periods (MANTELATTO & SOUSA, 2000;MIRANDA & MANTELATTO, 2009).Considering the symbiotic lifestyle and that A. aidae is not monogamous (D.F. Peiró, J. A. Baeza & F. L. Mantelatto, unpublished data) we cannot discard the hypothesis about the influence of the host aspects on the frequency distribution pattern observed here.
In some studies with Pinnotheridae -due to its typical symbiotic relationship -the main variables were expressed in number of individuals per host, per pipe aperture or per hosts' gallery aperture (MCDERMOTT, 1981;ALVES & PEZZUTO, 1998A;MCDERMOTT, 2006).Comparing our results with other Austinixa species, the number of host gallery apertures is a differential factor to be considered, once they vary among the Callianassidae members (Tab.II).
Space and food availability in the gallery may be the most important factors controlling the Pinnotheridae density.The strong currents and water changes produced by the host provide a high supply of oxygen and food at the burrow entrance, probably in a sufficient rate for many crabs (ALVES & PEZZUTO, 1998a).The maximum and the mean densities of A. aidae per gallery were lower than the values found in A. patagoniensis from the Southern Brazil (state of Rio Grande do Sul) associated with Callianassidae Sergio mirim (Rodrigues, 1971) (ALVES & PEZZUTO, 1998a), and higher than those found in A. gorei associated with Callianassidae Gilvossius setimanus (DeKay, 1844) from Miami (USA) (MCDERMOTT, 2006) (Tab.III).Again, as mentioned above, we cannot discard the hypothesis about the influence of the host aspects on the pattern observed here.
The total density increases were directly influenced by the recruitment and/or entrance of juveniles in the population (mainly in May and in July); the increases of this interest group reflect significantly on the total population.Differently from the Pinnotheridae that live inside the shells, on the echinoderms, or in relatively small polychaets' pipes (MANNING & MORTON, 1987;ALVES & PEZZUTO, 1998A;CAMPOS, 1990), the galleries of C. major provide a sufficient space to the coexistence of a higher number of A. aidae, as found here (1.72 ± 1.34 ind.• ap. - ).The mean incidence of Austinixa is among the highest when compared with other Pinnotheridae species (ALVES & PEZZUTO, 1998a).However, A. aidae owns the lowest mean incidence among the three studied species of the genus, 75.6% (Tab.III).This parameter shows strong variation among the Pinnotheridae species, and more studies are necessary for better conclusions.The high incidence in samples from autumn and winter months corresponds to recruitment.The correspondence between the males' and females' density was more evident after the juveniles' peaks.Decrease in this parameter was observed after the peaks.It is important to register the strong drop in the incidence and in the number of collected individuals in January, 2006, which followed the decrease that started bimonthly before, in November, 2005.That period corresponded to a higher incidence of tourists in the area using suction pumps to capture the host and use it in non-professional fisheries.Although no studies are available to check this matter, and analogously to what was observed in the southern area (SOUZA & BORZONE, 2003), we believe in the influence of those practices on the dynamics of both host and pinnotherid populations in this area during the summer.The analysis between the density of A. aidae and the size of their respective host shows a positive correlation, as opposed to what was found in P. patagoniensis when associated with Sergio mirim (ALVES & PEZZUTO, 1998a).This result grants a peculiar characteristic to A. aidae in the family.The positive correlation is more frequently observed among crabs that are symbiotic of bivalves or polychaets tubes, where the free space in the shell is limited (GRAY, 1961;PEARCE, 1966A, 1966B;JONES, 1977;BIERBAUM & FERSON, 1986).
The sex ratio did not differ significantly from the pattern proposed by FISCHER (1930), where the natural selection supports an equal proportion for both sexes.In most of the sampled months, the sex ratio was in favor of females, yet not significantly different from 1:1 (p < 0.05).When a ratio variation occurs, it can be explained by mortality, habitat preferences and alimentary restrictions acting intensively on one sex (TURRA & LEITE, 2000); however, these factors do not evaluated here.In addition, the previous hypothesis that suggested that adult crabs featured a mating system other than monogamy in the studied species (D.F. Peiró, J. A. Baeza & F. L. Mantelatto, unpublished data) should be considered an explanation for the pattern of the sex ratio observed here.
It was not possible to apply any classification proposed by WENNER (1972) to the sex ratio pattern in this population.No significant differences between the male-female ratios in the majority of size classes were found; however, there were significant differences in the 2 nd and the 9 th size classes in favor of males and in the 7 th in favor of females.This probably brings us to a new classification that has been registered here for this group of symbiont and that we named "near pattern".This classification is close to the "pattern" proposed by WENNER (1972), in which there are no differences in the male/female proportion in most size classes, but allows some few significant differences between the sexes, in favor of males in the final size classes.
The sex ratio of A. aidae is similar to that found in A. patagoniensis ( 1:1) (ALVES & PEZZUTO, 1998a), however it diverges significantly from A. gorei (0.  1981).These traits may reflect adaptations between hosts, species and environmental conditions, demonstrating a high plasticity of the species' sex ratio features in their distribution.
Reproductive period and recruitment.The ovigerous females of A. aidae were present throughout the year; however, they were more expressive in May and in November, 2005 andin May, 2006.The reproductive pattern at Perequê-açu beach was classified as Seasonal Continuous (according to PINHEIRO & TERCEIRO, 2000), corroborating the pattern found in the congener A. patagoniensis (ALVES & PEZZUTO, 1998a) NEGREIROS-FRANSOZO, 1999;MANTELATTO & FRANSOZO, 1999;PINHEIRO & TERCEIRO, 2000, respectively).Generally, Brachyura crustaceans from temperate areas present seasonal reproduction, which occurs in the hottest seasons of the year (WARNER, 1977;ASAKURA & KIKUCHI, 1984), while crabs from tropical and subtropical regions have a continuous reproduction throughout the year, usually in approximate rates (SASTRY, 1983).
Comparing this study to others realized with the congeneric A. patagoniensis from southern Brazil, and with A. gorei from Florida (USA), similar patterns were verified in ecological and biological aspects among the three species, even in cases from far geographical localities, corroborating the postulated by other authors who had referred the similarity of these parameters among the Austinixa genus (MANNING & FELDER, 1989;HEARD & MANNING, 1997;ALVES & PEZZUTO, 1998A;MCDERMOTT, 2006;D. F. Peiró, J. A. Baeza & F. L. Mantelatto, unpublished data).However, a comparison of the A. aidae population dynamics with other species of pinnotherids was not entirely satisfactory due to a lack of information and studies about their geographical distribution limits.The differences regarding the relation with the host size obtained between A. aidae and C. major inspire new ideas and doubts about this interesting lifestyle that should be evaluated in the future in order to better contribute to the understanding of the symbiosis evolution.