Food Restrictions Affect the Larval Metamorphosis and Early Juvenile Performance in a Neotropical Mangrove Fiddler Crab (Leptuca cumulanta).
During their development, planktonic larvae of many benthic marine invertebrates may experience extreme variations in environmental factors (Anger, 2001; Calado and Leal, 2015), such as temperature, salinity, predation risk, and unpredictable periods of food availability (Rumrill, 1990; Wehrtmann, 1991; Morgan, 1995; Anger, 2001). With the exception of lecithotrophic larvae, which develop in the absence of food, most larval stages of decapod crustaceans depend on the availability of exogenous food sources (i.e., planktotrophic; Anger, 2001); and they may be affected biologically and ecologically by the variation in nutritional conditions (Allison, 1994). Periodic scarcity of nutrients may, therefore, affect the survival, developmental period, and growth of decapod larvae (McConaugha, 1982; Dawirs, 1987; Mikami et al., 1995; Gimenez, 2002; Gimenez and Anger, 2005; Rotllant et al., 2010; Guerao et al., 2012), which may have a direct impact on the success of larval recruitment (Olson and Olson, 1989). In decapod species, the detrimental effects of food shortage and nutritional vulnerability have been evaluated primarily during different periods of larval starvation or feeding through the determination of the point of no return (PNR) and the point of reserve saturation (PRS). The PNR is the period of initial starvation that inhibits the survival of the larvae, even after subsequent feeding, while the PRS is the minimum period of initial feeding by the larvae that permits their subsequent development, irrespective of the availability of food (see Anger and Dawirs, 1981; Anger, 1987, 2001; Gebauer et al., 2010). The effects of food scarcity may nevertheless have different implications for the larvae of different species (Freeman, 1990; Mikami et al., 1995), whether at the beginning or toward the end of their meroplanktonic development (Calado et al., 2010). The influence of nutritional stress (or different periods of food deprivation; Gimenez, 2002) on the early larval stages of decapod crustaceans has been described in a number of different studies (e.g., Paul and Paul, 1980; Anger et al., 1981, 1985; Staton and Sulkin, 1991; Abrunhosa and Kittaka, 1997; Paschke et al., 2004; Gebauer et al., 2010; Guerao et al., 2012; Barros-Alvez et al., 2018). However, the impacts of transitory periods of food limitation have been little investigated in the last larval stage (i.e., megalopa) of this invertebrate group (e.g., Farrelly and Sulkin, 1988; Harris and Sulkin, 2005; Holme et al., 2009; Souza et al., 2017).
In addition to the effects on larvae, stressful conditions (e.g., food limitation, low salinity, toxic pollutants, and prolonged developmental period) experienced early in ontogeny (e.g., embryonic or larval phase) may also affect the postmetamorphic performance of early juveniles and/or adults (Hunt and Scheibling. 1997; Gimenez et al., 2004; Anger, 2006; Gimenez, 2006; Pechenik, 2006; Podolsky and Moran, 2006; Simith et al., 2013). This biological link with the prior embryonic or larval history may transmit phenotypic effects (commonly referred to as "carryover" or "latent" effects) and impact survival, development, morphology, and physiology of the juveniles, as observed in a number of different benthic marine invertebrates (see Pechenik et al., 1998; Pechenik, 2006). These latent effects emphasize the idea that "metamorphosis is not a new beginning" for these organisms (reviewed by Pechenik et al., 1998; Pechenik, 2006). For example, some studies have shown that short-term and/or long-term periods of food deprivation in larvae may significantly affect early juvenile development (Allison, 1994; Zheng et al., 2005). Limited feeding or short periods of starvation during larval life may result in a significant reduction in weight, body size, and growth rates of juveniles (see Miller, 1993; Phillips, 2002; Emlet and Sadro, 2006). Such effects have been observed in the gastropods Crepidula fornicata (Pechenik et al., 1996a, b, 2002; Pechenik and Tyrell, 2015) and Crepidula onyx (Chiu et al., 2007), the bivalve Mytilus galloprovincialis (Phillips, 2002, 2004), the polychaete Polydora ligni (Qian et al., 1990), and the barnacles Balanus amphitrite (Thiyagarajan et al., 2003) and Austrominius modestus (Torres et al., 2016). The carryover effects resulting from the different periods of food limitation on early post-metamorphic development have rarely been investigated in decapod crustaceans. For example, a reduced body size of recently metamorphosed juveniles (i.e., first crab stage) was evidenced in the ornamental red-ridged clinging crab, Mithraculus forceps, when megalopae experienced a starvation regime during the final phase of their meroplanktonic development (see Figueiredo et al., 2008). Recently, Rey et al. (2016) also showed that unfavorable feeding conditions occurring during the preceding larval phase may affect early juvenile growth in the European green crab, Carcinus maenas.
The mangrove fiddler crab Leptuca cumulanta (Crane, 1943) is a common semi-terrestrial species living in the intertidal zone of tropical and subtropical areas of the western Atlantic (Crane, 1975; Koch et al. 2005), between Central America and Brazil, where it ranges from the state of Para to the state of Rio de Janeiro (Melo, 1996; Thurman et al., 2013). Leptuca cumulanta has four zoeal stages and one megalopal stage (TNR, ASdeS, FAA, DJBS, M. A. B. Pires, Dickinson College, unpubl. data). The number of zoeal stages characterizes the mode of development of this species as intermediate, between abbreviated, with two or three partially or totally lecithotrophic stages, and prolonged, with five or more planktotrophic stages (Gore, 1985; Anger, 2001; Anger et al., 2015). The few studies on this species have examined the asymmetry of the chelipeds, growth, sexual maturity, behavior, morphology of the juvenile stages, and population dynamics (see Ahmed. 1978; Chiussi and Diaz, 2001; Koch et al., 2005; Pralon and Negreiros-Fransozo, 2008; Hirose et al., 2010). However, the degree of nutritional vulnerability and the influence of the nutritional history of the megalopal stage on the development of early benthic juvenile recruits have yet to be investigated in L. cumulanta.
In the present study, we investigated, in two laboratory experiments, whether different periods (1,3, and 5 days) of initial starvation (PNR experiment) or initial feeding (PRS experiment) affect the survival (i.e., % larval metamorphosis to juvenile) and the duration of the megalopal stage of this species. We also assessed whether the survival, growth, intermolt period, body size, and shape of the first 5 juvenile (JI-JV) crab stages of L. cumulanta are altered when they experience 2 different periods (I and 3 days) of initial starvation during their megalopal life. In addition, we used the Nutritional Vulnerability Index (NVI) (see Gebauer et al., 2010) to determine the degree of dependence of the megalopae of L. cumulanta on external food sources during their development until the metamorphosis to juvenile.
Materials and Methods
Culture medium acquisition and treatment
The seawater for larval culture of Leptuca cumulanta (Crane, 1943) was collected from the coastal region in northern Brazil (0[degrees]49']5.7" S, 46[degrees]36'23.46" W). This water was filtered through a 5-[micro]m mesh screen and stored in 500-L tanks in the laboratory. Two biological filters (i.e., biofilters) were placed within each 500-L tank to keep the ammonia and nitrite concentrations low (see Valenti et al., 1998, 2009, 2010; New, 2002; Timmons et al., 2002). The seawater was then filtered under constant aeration, using these biofilters for up to 30 days before the larval culture. The biological filters consisted of calcareous material (e.g., bivalve shells) that provided a substratum for the growth of nitrifying bacteria. These filters have been used to keep the water quality favorable for decapod larvae culture (see Valenti et al., 2010, and references cited therein). Further infonnation on the biofilters can be found in Valenti et al. (1998, 2009, 2010), New (2002), and Timmons et al. (2002).
Larval acquisition and culture conditions
Ovigerous females of L cumulanta (n = 4; carapace width = 0.73 [+ or -] 0.11 cm [[+ or -]SD]) were collected 2 days prior to spawning in the mangroves of the Caete River estuary, in northern Brazil (00[degrees]50'33.62" S, 46[degrees]38' 17.29" W). In the laboratory, each female was placed in a 2-L separate aquarium containing filtered seawater with salinity = 30, mean water temperature = 27 [+ or -] 0.5 [degrees]C, and pH = 8.1; and all were maintained with a 12h : 12h fight: dark photoperiod regime until the larval release. Once hatched, only the actively swimming zoea larvae (displaying a positive phototropism) produced by the different females were collected and mixed in a 500-mL glass beaker, to avoid possible influence of genetic differences and/or parental conditions (e.g., age, health, physiological and nutritional state, diseases) on the performance of larvae and juveniles in the experiments. Thereafter, these larvae were transferred randomly to 230-mL plastic containers, where they were cultured at a density of about 40 larvae per container under the same conditions of salinity, temperature, pH, and photoperiod as the adult females. The larvae were fed daily with microalgae (Nannochloropsis sp.) at a concentration of about 1 X [10.sup.-] cells m[L.sup.-1] and with marine rotifers (Brachionus plicatilis) at a density of about 90 rotifers m[L.sup.-1]. Recently hatched brine shrimp (Anemia sp.) nauplii were also provided (at a density of about 6 nauplii m[L.sup.-1]) from the third zoeal stage (ZIII) onward until larvae reached the megalopal stage. The culture medium was renewed every two days, and the food was added soon after. After the larval release, the female crabs were returned to the mangroves.
Experimental design for megalopal culture
Once larvaae had reached the megalopal stage, they were assigned to one of two experiments; PNR (point of no return; Fig. 1A) or PRS (point of reserve saturation; Fig. 1B). Prior to the culture in the PNR experiment, the digestive system of each recently molted megalopa was carefully examined for previous ingestion of food. In this procedure, the megalopae were collected with wide bore pipettes, placed in a drop of seawater on a glass slide, and examined under a stereoscopic microscope (Axioskop 40, Zeiss, San Diego, CA) for up to 5 s to avoid stress and desiccation. Only megalopae with empty (i.e., translucent) stomachs and guts were retained for this experiment. These megalopae were then subjected to 1 of 3 experimental treatments with 3 different periods (1,3, and 5 days) of initial starvation (hereafter referred to as starvation-treatments S1, S3, and S5, respectively), followed by a continuous period of feeding (Fig. 1 A). The PRS experiment also involved 3 experimental treatments with 3 different periods (1,3, and 5 days) of initial feeding (hereafter feeding-treatments Fl, F3, and F5, respectively), followed by a constant period of food deprivation (Fig. 1B). Two different control groups were also established, in which the megalopae were starved (starvation control [SC]) or fed (feeding control [FC]) continuously. Only one starvation control and one feeding control were conducted and used for multiple and pairwise comparisons in both experiments.
The age of the megalopae (counted from the day of hatching to the day of molting to the megalopal stage) varied from 9 to 20 days (13.5 [+ or -] 3.5 days [[+ or -]SD]). Megalopae that presented different durations of the zoeal phase were therefore distributed at the same proportions among the different treatments and control groups of each experiment (PNR and PRS). By distributing megalopae of different ages equally among the treatments, the possible effects that a short or a long zoeal developmental period may have on the health, survival, and/or megalopal stage duration of L. cumulanta could be reduced or avoided in our experiments. Each treatment and control group initially had 40 megalopae that were cultured individually in 170-mL plastic containers. Each megalopa was therefore considered an experimental replicate within each treatment (see Forward et al., 2001). In both experiments, an empty marine bivalve shell was placed in all the containers belonging to each treatment and control as a natural stimulus for the induction of metamorphosis of the megalopae of L cumulanta. Each shell was washed, brushed, and autoclaved in a CS vertical autoclave (Prismatec, Sao Paulo, Brazil) prior to use. The presence of a mollusc shell (i.e., physical stimuli) in the culture should prevent negative effects (e.g., reduced survival and lengthening of the megalopal stage duration) caused by the absence of metamorphosis-stimulating cues during the larval competence period of decapod species (see review in Anger, 2001; Forward et ah, 2001; Gebauer et al., 2003).
The megalopae were fed exclusively on recently hatched Artemia sp. nauplii (at a density of about 2 nauplii m[L.sup.-1]) according to the different periods of addition (PNR experiment) or withdrawal (PRS experiment) of food (see Fig. 1 A, B). The abiotic culture conditions (temperature, salinity, pH, and photoperiod) were the same as those during the culture of the zoeal stages. The culture medium was renewed every two days. In both experiments, the megalopae were monitored daily for mortality and/or metamorphosis to the first juvenile (JI) crab stage. The experiments were terminated when the last megalopae had either reached the JI crab stage or died in the treatment or control groups.
Juvenile culture and quantification of the post-metamorphic performance
We used juvenile crabs developed from megalopae cultured in starvation-treatments S1 and S3, together with those specimens produced by the megalopae fed continuously in the FC group, to investigate whether the nutritional history of the preceding larval phase affected the early post-larval development of L. cumulanta (Fig. 1C). The individual crabs were cultured separately in 170-mL plastic containers through the first 5 juvenile (JI-JV) stages. Each treatment (FC, S1, and S3) initially included 30 recently metamorphosed specimens in the JI crab stage. To complete the initial sample size of starvation-treatment S3, we cultured the remaining megalopae produced by the different females of origin under the same conditions as those described for the PNR experiment (see Experimental design for megalopal culture). The juveniles were then cultured in seawater with a mean temperature = 27.0 [+ or -] 0.5 [degrees]C ([+ or -]SD), salinity = 30, and pH = 8.0, with a 12h : 12h light: dark photoperiod cycle. The water in the containers was renewed every two days. The crabs were fed daily ad libitum with recently hatched Artemia sp. nauplii (at a density of 6-10 nauplii m[L.sup.-1]), and they were examined for mortality or molt events to the subsequent juvenile stages. The experiment was ended when the last specimen had either molted to the fifth juvenile (JV) crab stage or died in each treatment.
After the molt events, the exuviae of each juvenile (JI-JIV) crab stage were collected and preserved in 70% ethanol +glycerin (1:1) solution for later measurement of carapace width (CW) and carapace length (CL), using a SMZ 168 stereomicroscope (Motic, Hong Kong) equipped with a calibrated micrometric eyepiece. For some crab stages, the total sample size for CW and/or CL was reduced because the exuviae of some individuals were not measured. This occurred because the carapaces were damaged or completely eaten by the crabs themselves. For JV crabs, live individuals were measured. The data for CW were used to calculate juvenile growth quantified as percentage of size increment at each consecutive molt (% SIM), using the following formula (see Lopez and Rodriguez, 1998; Simith et al., 2013):
[mathematical expression not reproducible] (1)
In the present study, the early juvenile performance of L. cumulanta was quantified based on biological parameters such as percentage survival, intermolt developmental period (in days), body size (CW and CL, in mm), growth (% SIM), and body shape (CL/CW).
Data analysis and statistical procedures
In both experiments, the values (in days) of the [PNR.sub.50] and [PRS.sub.50] indexes, which are based on the time interval during which 50% of the megalopae either reached the PNR or the PRS, respectively (see Anger, 1987; Paschke et al., 2004; Bas et al., 2008; Gebauer et al., 2010), were estimated using the percentage of larval survival (i.e., % of metamorphosis to JI crab stage) recorded in the different treatments and control groups (SC and FC). The values of the [PNR.sub.50] and [PRS.sub.50] indexes were then determined by adjusting the sigmoidal Boltzmann model to the least squares approach, using the following equation (for applications see Gebauer et al., 2010; Guerao et al., 2012; Souza et al., 2017, 2018):
[mathematical expression not reproducible] (2)
where y = percentage of larval metamorphosis to the JI crab stage recorded in each treatment (PNR = S1, S3, and S5; PRS = F1, F3, and F5) and control group (SC and FC); a = the highest percentage of larval metamorphosis; b = the value of [PNR.sub.50] or [PRS.sub.50] in days; c = the slope of the curve; and X = the respective periods (1,3, and 5 days) of initial starvation or initial feeding of each experiment (PNR or PRS, respectively). Because the percentages of larval metamorphosis recorded in the control groups (SC and FC) were also used in the component y of the equation above, these respective treatments had to be included to fit the slope of the curve. Therefore, for these control groups we assigned a value of 7 days, following the sequence of the periods of starvation or feeding plotted in intervals of 2 days (1, 3, and 5 days) and used in the component x. By assigning periods of 7 days onward for SC and FC, the slope of the survival curve became stable, and the estimated values of the [PNR.sub.50] and [PRS.sub.50] indexes became constant. The NVI was estimated using the following formula (Gebauer et al., 2010):
[mathematical expression not reproducible], (3)
with values of NVI [less than or equal to] 0.5 indicating a low degree of dependence on exogenous food (i.e., low nutritional vulnerability), values of NVI between 0.5 and 1.0 indicating an intermediate level of dependence on food supply, and values of NVI [greater than or equal to] 1.0 indicating a high degree of dependence on external food sources (i.e., high nutritional vulnerability) to complete the development of the megalopae through metamorphosis into the JI crab stage.
The effects of the different treatments on the percentage survival of the megalopae and juveniles (JI-JIV) were analyzed through contingency tables (rows vs. columns) followed by the chi-square test ([chi square]) with Yates' correction. The data on the duration of the megalopal stage until metamorphosis to the JI crab stage (hereafter referred to as megalopal stage duration [MSD], in days), as well as the intemolt period, body size (CW and CL), body shape (CL/CW), and absolute SIM of juvenile crabs, were compared among the different treatments using a one-way analysis of variance (ANOVA). Prior to this analysis, the normality and homogeneity of the variances of these data were verified using Kolmogorov-Smimov's and Levene's tests, respectively. Whenever necessary, the data were ([x.sup.1/2]) + [([x.sup.1/2]) + 1] transformed. When significant differences were detected through the ANOVA, Tukey's honest significant difference (HSD) test was applied to pairwise comparisons between the experimental treatments (S1, S3, and S5 or Fl, F3, and F5) and between these treatments and the FC group. Pearson's correlation coefficient (r) was calculated to verify the relationship between the data on MSD and the intermolt period of the JI crab stage cultured in treatments S1, S3, and FC. Differences were considered to be significant when P values were <0.05 of the significance level. The data were presented as mean [+ or -] SD values.
PNR experiment: effects of different periods of initial starvation
In the first experiment, the continuous regime of starvation (SC) resulted in total mortality of the megalopae of Leptuca cumulanta after 3-12 days (7.6 [+ or -] 2.7 days to death), whereas 80% of the megalopae metamorphosed to the first juvenile (JI) crab stage when fed continuously in the FC group. In general, survival of the megalopae decreased as the period of initial starvation (1,3, and 5 days) increased (Fig. 2A). With only 1 day of starvation (starvation-treatment S1), the percentage of metamorphosed megalopae did not differ significantly ([chi square] = 0.55, P = 0.45) from that recorded in the FC group (Fig. 2A). By contrast, periods of 3 days (S3; [chi square] = 13.13, P < 0.01) or 5 days (S5; [chi square] = 48.51, P < 0.01) of initial starvation significantly reduced the percentages of larval metamorphosis in comparison with treatments FC and S1, respectively (Fig. 2A).
In this experiment, the megalopal stage lasted from 6 to 25 days. The mean development duration of the megalopae of L. cumulanta until reaching metamorphosis to the JI crab stage (i.e., MSD) differed significantly (ANOVA, [F.sub.3,97] = 5.86, P < 0.01) between the different treatments (S1, S3, S5, and FC; Fig. 2B). The mean MSD was similar (Tukey's HSD test, P > 0.05) between starvation-treatments S1, S3, and S5 (12.6 [+ or -] 3.5 days; pooled data). However, when the larvae were initially starved for 1, 3, or 5 days, the mean MSD was 2.2, 3.3, or 3.7 days significantly longer, respectively (Tukey's HSD test, P < 0.05), compared to those larvae fed continuously in the FC group (10.6 [+ or -] 2.4 days; Fig. 2B).
PRS experiment: effects of different periods of initial feeding
The different periods (1, 3, or 5 days) of initial feeding (feeding-treatments Fl, F3, or F5, respectively) also affected the percentage of larval metamorphosis in L. cumulanta (Fig. 2D). The larvae cultured with only a single day of feeding (Fl) had the lowest percentage of metamorphosis to the JI crab stage (15%) in comparison with those larvae fed for 3 days (F3; [chi square] = 38.05, P < 0.01) and 5 days (F5; [chi square] = 46.43, P < 0.01) or constandy in the FC group ([chi square] = 73.49, P < 0.01; Fig. 2D). Furthermore, the percentages of metamorphosis recorded in F3 and F5 were also significantly lower (F3: [chi square] = 10.31, P < 0.05; F5: [chi square] = 6.28, P< 0.05) than that observed in FC (Fig. 2D).
The MSD of L. cumulanta ranged from 5 to 19 days in this experiment. In contrast to larval survival, the mean MSD was not significantly different (ANOVA, [F.sub.2.74] = 0.43, P = 0.65) between the treatments with 3 and 5 days of initial feeding (F3 and F5, respectively) and the FC group (Fig. 2E). In these treatments, the larvae exhibited a mean MSD of 10.4 [+ or -] 2.3 days (pooled data). In addition, while feeding-treatment F1 was not included in the analysis due to the small sample size, its mean MSD was 1.9 days shorter than that of the FC group (Fig. 2E).
Nutritional vulnerability index
In the PNR experiment, the estimated value of [PNR.sub.50] was 3.9 days (Fig. 2C), while in the PRS experiment, [PRS.sub.50] was estimated at 2.4 days for the megalopae of L. cumulanta (Fig. 2F). The [PRS.sub.50] : [PNR.sub.50] ratio thus provided an NVI of 0.62, which is consistent with an intermediate degree of dependence on exogenous food during the development of the megalopae until metamorphosing to the JI crab stage.
Carryover effects of larval starvation on early juvenile performance
Survival of early juvenile (JI-JIV) crab stages of L. cumulanta was not affected by the different periods (1 and 3 days) of initial starvation experienced early during the megalopal stage. The percentages of survival of the JI-JIV crab stages were high (>90%) in both the starvation treatments (S1 and S3) and the FC group. In starvation-treatment S1, 100% survival was recorded for the JI and JIII crab stages, 95% for JII, and 92% for JIV, while in starvation-treatment S3, survival was 93% for the JI stage and 100% for the other juvenile stages (JII-JIV). In the FC group, survival was 97% for the JI stage and 100% for the remaining crab stages (JII-JIV).
The intermolt period increased during the course of juvenile development (from JI to JIV) of L. cumulanta, but it varied in the different treatments where the megalopae were initially starved for 1 and 3 days or fed continuously (Fig. 3A). The mean total duration from the day of metamorphosis to the first juvenile (JI) crab stage until the day of molting to the last juvenile crab stage (JV) varied from 19 to 87 days (35.3 [+ or -] 14.3 days) in S1, from 19 to 72 days (32.3 [+ or -] 11.4 days) in S3, and from 18 to 65 days (41.5 [+ or -] 14.4 days) in FC. The mean intermolt developmental period for the JI stage varied significantly (ANOVA, [F.sub.2,78] = 7.7, P < 0.05) between treatments SI, S3, and FC (Fig. 3A). The JI crab stage exhibited an intermolt period 1.6 or 3 days shorter (Tukey's HSD test, P < 0.05) in S3 than in SI or FC, respectively (Fig. 3A). Pearson's coefficient (r) showed that the correlation between the MSDs and the intermolt periods of the juveniles (JI) was not statistically significant in the respective treatments S1 (r = 0.13; t = 0.67; P = 0.51), S3 (r = -0.35; t= -1.11; P = 0.09), and FC (r = -0.06; t = -0.31; P = 0.76). In the subsequent juvenile crab stages (JII-JIV), the mean intermolt period did not vary significantly between treatments S1, S3, and FC (pooled data; JII = 6.0 [+ or -] 2.1 days, ANOVA, [F.sub.2,84] = 0.42, P = 0.66; JIII = 8.6 [+ or -] 4.2 days, ANOVA, [F.sub.2,81] = 0.65, P = 0.53; JIV = 12.4[+ or -]4.8 days, ANOVA, [F.sub.2,81], = 1.79, P = 0.17; Fig. 3A).
The different periods of initial starvation (1 and 3 days) experienced by the megalopae also affected the mean body size (CW and/or CL) of the early juvenile (JI-JIII or JV) crab stages of L. cumulanta (Fig. 3B, C). In the JI crab stage, mean CW varied significantly (ANOVA, [F.sub.2,76] = 4.41, P < 0.05) among treatments and was 6% or 8% significantly smaller (Tukey's HSD test, P < 0.05) in starvation-treatment S3 in comparison with treatments FC or S1, respectively (Fig. 3B). The mean CW of the JII and JUI crab stages also differed significantly among treatments (JII: ANOVA, [F.sub.2,83] = 13.96, P < 0.01; JIII: ANOVA, [F.sub.2,86] = 6.78, P < 0.01; Fig. 3B). The mean CW of the JII and JIII crab stages was 9.4% and 7.2% significantly greater (Tukey's HSD test, P < 0.05) in S1 than in S3 or FC, respectively (Fig. 3B). For JIV crabs, we found no significant variation (ANOVA, [F.sub.2,87] = 0.68, P = 0.51) in the mean CW (1.6 [+ or -] 0.2 mm; pooled data) between treatments S1, S3, and FC (Fig. 3B). By contrast, the mean CW of specimens in the JV crab stage differed significantly (ANOVA, [F.sub.2,85] = 9.81, P < 0.05) among treatments and was 9.6% and 11.5% significantly larger (Tukey's HSD test, P < 0.05) in S3 when compared to treatments S1 and FC, respectively (Fig. 3B).
The pattern of variation in CL of the first three juvenile (JI-JIII) crab stages was similar to that recorded for CW (Fig. 3C). For the JI crab stage, mean CL varied significantly among treatments (ANOVA, [F.sub.2,79] = 3.13, P < 0.05), being 14.3% smaller in starvation-treatment S3 (Tukey's HSD test, P < 0.05) in comparison with treatment S1 or the FC group (Fig. 3C). In the JII crab stage, mean CL was also significantly different (ANOVA, [F.sub.2,80] = 16.83, P < 0.01), being 9.4% larger in starvation-treatment S1 (Tukey's HSD test, P < 0.05) in comparison with treatments S3 or FC (Fig. 3C). The mean CL of the JIII crab stage differed significantly between treatments (ANOVA, [F.sub.2,83] = 7.28, P < 0.01), with the juveniles cultured in S1 being 5.9% larger (Tukey's HSD test, P < 0.05) in comparison with the specimens of treatment S3 and the FC group (Fig. 3C). In contrast to the former juvenile stages, the mean CL of JIV and JV did not vary significantly between treatments S1, S3, and FC (pooled data; JIV = 1.6 [+ or -] 0.1 mm, ANOVA, [F.sub.2,87] = 2.48, P = 0.09; JV = 1.8 [+ or -] 0.1 mm, ANOVA, [F.sub.2,85] = 0.18, P = 0.84; Fig. 3C).
No significant variation between treatments S1, S3, and FC was found in body shape (CL/CW) of the early juvenile (JI-JV) crab stages of L. cumulanta (JI: ANOVA, [F.sub.2,73] = 2.36, P = 0.1; JII: ANOVA, [F.sub.2,83] = 1.14, P = 0.32; JIII: ANOVA, [F.sub.2,82] = 1.02, P = 0.36; JIV: ANOVA, [F.sub.2,87] = 1.57, P = 0.21; JV: ANOVA, [F.sub.2,85] = 2.55, P = 0.84). The analysis of the body shape (CL/CW) of juveniles showed that the carapace is slightly longer than it is wide in the JI (1.16 [+ or -] 0.09), JII (1.09 [+ or -] 0.05), JIII (1.05 [+ or -] 0.07), and JIV (1.04 [+ or -] 0.05) crab stages. After molting to the JV crab stage, the carapace became slightly wider than longer (CL/CW = 0.94 [+ or -] 0.06).
Early juvenile growth, measured by % SIM, varied significantly between treatments S1, S3, and FC for most molt events (Fig. 3D). The mean % SIM of the first molt event (I-II) differed significantly among treatments (ANOVA, [F.sub.2,71] = 10.59, P < 0.05; Fig. 3D). After this molt event (I-II), the size increment was 21.4% greater in the juveniles produced by the megalopae cultured in starvation-treatment S1 (Tukey's HSD test, P < 0.05) compared to those specimens obtained from megalopae cultured in treatments S3 and FC (Fig. 3D). After the second molt event (II-III), by contrast, the mean % SIM (22.2% [+ or -] 3.8%; pooled data) did not vary significantiy among treatments (ANOVA, [F.sub.2,76] = 0.07, P = 0.94; Fig. 3D). After the third molt event (III-IV), however, a significant variation (ANOVA. [F.sub.2,74] = 22.71, P < 0.01) was found in the % SIM in treatments S1, S3, and FC (Fig. 3D). Juvenile crabs cultured in starvation-treatment S1 had a mean % SIM 38.3% lower than the specimens from treatments S3 and FC (Fig. 3D). Significant variation (ANOVA, [F.sub.2,78] = 25.08, P < 0.01) was also found after the fourth molt event (IV-V), with juvenile size increment in starvation-treatment S3 being 40.6% higher (Tukey's HSD test, P < 0.05) than in the other treatments (Fig. 3D).
The results of the present study demonstrated that the megalopae of the mangrove fiddler crab Leptuca cumulanta are unable to tolerate complete starvation, dying after a mean of 7.6 days without food. Our study also showed that different regimes (1, 3, or 5 days) of initial starvation (FNR experiment) may significantly reduce the percentage of larval survival and extend the mean megalopal stage duration (MSD) until metamorphosis to the first juvenile (JI) crab stage of this species. Consequences of temporary food limitations on the megalopal stage have also been observed in other brachyuran species (see Farrelly and Sulkin, 1988; Harris and Sulkin, 2005; Figueiredo et al., 2008; Holme et al., 2009; Souza et al., 2017, 2018). In L. cumulanta, a significant reduction in the number of surviving larvae was observed when their megalopae were initially starved for 3 or 5 days, while a significant increase of the MSD (by 21%, to 35%) occurred irrespective of the starvation period, compared to continuously fed larvae (FC group).
In many decapod crustaceans, the larval period is extended in the absence of planktonic food and/or environmental stimuli associated with the habitat of the conspecific population (Anger, 2001; Forward et al., 2001; Gebauer et al., 2003; Souza et al., 2017). Alterations in the duration of the molting cycle have also been observed in the early zoeal stages of some crab species after they experience a starvation regime (e.g., the great spider crab, Hyas araneus: Anger and Dawirs, 1981; the white-fingered crab Rhithropanopeus harrisii: McConaugha, 1982; the fatfinger marsh crab Armases miersii: Anger, 1995a; the mangrove marsh crab Sesarma curacaoense: Anger. 1995b). In contrast with megalopae of other crab species (e.g., S. curacaoense, Sesarma rectum: Souza et al., 2017; the mangrove crab Ucides cordatus: Souza et ah, 2018), the extension of the megalopal period of L. cumulanta also occurred with only a single day of starvation. In general, short-term periods of starvation should not affect the developmental period because the larvae of most crustaceans may utilize their lipid reserves to satisfy nutritional requirements during transitory periods of food scarcity (Narciso and Morals, 2001; Smith et al., 2003). The impact of only one day of starvation on the megalopae of L. cumulanta could also be caused by the physiological and/or nutritional state of the preceding zoeal stages (see Holme et al., 2009; Guerao et al., 2012) and/or stressful environmental conditions experienced early during embryonic development (see Gimenez and Anger, 2001, 2003). It is not possible to know whether the larvae produced by the different females of L. cumulanta have faced unfavorable conditions during their embryogenesis in the field. Because the zoea larvae in our study were fed at the same frequency, concentration, and type of food, the observed effects were likely caused by the different conditions of starvation experienced by the megalopae instead of the previous nutritional history of the zoeae. While the total developmental time of the preceding zoeal phase could also affect the MSD, this did not affect our study because the older and younger megalopae were distributed evenly among the treatments and control groups. Our findings, therefore, corroborate those reported in the literature and indicate that the timing of starvation may be as important as its duration within the development of the megalopal stage of decapod species (Anger and Dawirs, 1981; Anger, 1984; Guerao et al., 2012; Souza et al., 2017, 2018).
The PRS experiment showed that just one day of initial feeding followed by complete starvation may cause a great reduction in the percentage of larval metamorphosis of L. cumulanta. When the feeding schedule was extended to three or five days, the percentage of larvae that metamorphosed to JI increased significantly when compared to the culture with only one day of feeding. The period in which 50% of the larvae reach PRS (i.e., [PRS.sub.50]; Anger, 1987; Paschke et al., 2004; Bas et al., 2008; Gebauer et al., 2010) was estimated at 2.4 days. This [PRS.sub.50] value was higher than that recorded for the megalopal stage of other crab species (e.g., 0.9 days in S. curacaoense, 1.8 days in S. rectum: Souza et al., 2017; 2.1 days in U. cordatus: Souza et al., 2018). This indicates that the megalopae of L. cumulanta are less able to tolerate the absence of food after experiencing short-term periods of feeding at the beginning of their development. Similar results have also been reported for the megalopae of the blackclaw crestleg crab, Lophopanopeus hellus. and the grooved mussel crab, Fabia subquadrata (Harris and Sulkin, 2005), which require periods from 6 to 8 days, respectively, of feeding (32.3%-58.4% of the mean duration of the megalopae fed continuously) to metamorphose to juvenile. In megalopae of L. cumulanta, however, the different periods of initial feeding (e.g., 3 or 5 days) did not significantly impact the mean MSD compared to the continuously fed control larvae. The significantly lower survival recorded in F3 and F5 compared to the FC group may indicate that these treatments selected only the healthiest megalopae (57.5% and 62.5%, respectively) in such feeding conditions.
The PNR experiment demonstrated that megalopae of L. cumulanta are relatively intolerant of long periods of initial starvation, as indicated by the [PNR.sub.50] of 3.9 days, while the PRS experiment showed that this larval stage has a reduced capacity to adapt to starvation after experiencing short periods of initial feeding, as shown by the [PRS.sub.50] of 2.4 days. The [PRS.sub.50]/[PNR.sub.50]) ratio provided an NVI of 0.62 for L. cumulanta. Following Gebauer et al. (2010), values of NVI [less than or equal to] 0.5 indicate that larvae exhibit a low level of dependence on exogenous food (i.e., low nutritional vulnerability), while those values of NVI [greater than or equal to] 1.0 indicate that they are highly dependent on external food supply (i.e., high nutritional vulnerability) for completing their development to the juvenile crab stage. The value of NVI that we estimated for L. cumulanta indicates an intermediate degree (i.e., 0.5 < NVI < 1.0; Gebauer et al., 2010) of dependence of the megalopae on exogenous food sources. Although this index suggests that the megalopae have some energy reserves, their development may suffer negative effects under unstable environmental conditions (e.g., seasonal or diel variation in food availability) compared to other larval stages that have a low necessity for further food (i.e., low nutritional vulnerability = NVI [less than or equal to] 0.5; see Gebauer et al., 2010). Therefore, unfavorable trophic conditions may have important ecological implications on the process of larval recruitment, establishment, and maintenance of L. cumulanta populations, which may account for possible fluctuations in the abundance of juveniles and adults in coastal environments.
In our experimental study we demonstrated that the early juvenile performance of L. cumulanta may be affected when different periods (e.g., 1 or 3 days) of initial food deprivation are experienced as megalopae. These periods of starvation significantly prolonged the mean MSD. In many benthic marine invertebrates, the lengthening of the megalopal stage caused by the absence of adequate stimuli and/or lack of food may carry highly significant costs (e.g., reduced survival, slow growth, and prolonged intermolt period) for the postmetamorphic development and fitness of the early juvenile recruits (Pechenik el al, 1998; Qian and Pechenik, 1998; Gebauer et al., 1999, 2003; Pechenik, 1999, 2006; Phillips, 2002; Thiyagarajan et al. 2003; Gimenez el al, 2004; Emlet and Sadro, 2006; Podolsky and Moran, 2006; Simith et al., 2013; Pechenik and Tyrell, 2015). In the present study, the magnitude of the carryover effects of transitory larval starvation varied considerably among the biological parameters (e.g., intermolt period, body size, and growth) measured in the early juveniles. Here, the different periods (1 and 3 days) of megalopal starvation did not affect the survival of the juvenile crab stages. Also, juveniles in the JI crab stage had a shorter intermolt period in treatment S3 than those cultured in treatments S1 or FC. In addition, we did not observe any relationship between juveniles (e.g., JI crab stage) that had a short or a prolonged intermolt period and those megalopae that metamorphosed earlier or later in the culture. The intermolt developmental period of the juveniles of L cumulanta was therefore independent of the MSD in our study. The reduction of the intermolt period (i.e., acceleration of the molting cycle) of the JI crab stage in treatment S3 may reflect a shift in the developmental process as a response to the complex interactions between larval nutrition, molt, and morphogenesis (McConaugha, 1982). Despite the fact that the starvation condition of treatment S3 caused an increase in the mean MSD in L. cumulanta. it is likely that the most vigorous megalopae surviving into the juvenile stage may have contributed to the reduced intermolt period observed in the JI crab stage. This could be possible because the different experimental conditions of starvation may naturally select the strongest larvae and exclude the weakest individuals within a larval group. In this case, however, larvae of poor quality may still survive in favorable feeding conditions, such as in the FC group, while the strongest surviving megalopae are affected by experimental starvation, with the resulting effects, therefore, transferred later to the benthic stage after metamorphosis. In addition, juveniles produced by larvae from a specific female may have accounted for the observed results, indicating that the maternal and/or genetic conditions may have some influence on offspring quality.
Our results demonstrated that the juveniles in the JI crab stage of L. cumulanta derived from megalopae subjected to 3 days of initial starvation (treatment S3) were significantly smaller in size (i.e., CW and CL) than those whose megalopae were deprived of food for only 1 day (treatment S1) or fed continuously (FC group). It is likely that the subsequent feeding of the megalopae after 3 days of initial starvation was insufficient to bring about an increase in size after metamorphosis. In treatment S3, almost 50% of the megalopae of L cumulanta reached the critical PNR (Anger and Dawirs, 1981; Anger, 1987). Once the larva exceeds the PNR, the deleterious effects, such as the degradation of tissue and the exhaustion of nutrients, cannot be reversed, even after subsequent feeding (Anger and Dawirs, 1981; Anger, 1987; Freeman, 1990). Therefore, the small body size (i.e., CW and CL) of the JI crab stage from treatment S3 may have been caused by the consumption of the internal energetic reserves of the megalopae during the three days of food scarcity. Furthermore, the lengthening of the MSD may have resulted in an additional expenditure of energy utilized for swimming during the upstream migration to the mangroves (i.e., recruitment) and the subsequent search for a definitive benthic habitat for settlement close to the conspecific population.
Subsequent juvenile development may also be altered by phenotypic traits produced by unfavorable environmental conditions (e.g., salinity, temperature, and food availability) experienced during the preceding embryonic or larval phase (see Gimenez and Anger, 2001, 2003; Gebauer et al., 2003; Gimenez, 2004, 2006; Gimenez et al., 2004). Latent effects produced by the nutritional status of the larvae on the postmetamorphic performance of early benthic recruits have also been demonstrated in a number of invertebrate species (see Pechenik et al., 1996a, b; Emlet and Sadro, 2006; Allen and Marshall, 2010, 2013; Pechenik and Tyrell, 2015). In the context of these effects, the reduced size of the recently metamorphosed juveniles may have negative implications for the ecological interactions between benthic species (Gebauer et al., 1999,2003; Anger, 2006; Simith et al., 2013). For example, smaller juveniles may be less competitive in the acquisition of benthic resources, such as food and shelter (Hines, 1986; Gebauer et al., 1999, 2003; Simith et al., 2013), with a consequent increase in the risk of mortality (Pechenik and Tyrell, 2015). Also, smaller juveniles may be more susceptible to inter- and intraspecific predation (i.e., cannibalism) either within or between groups of individuals that have recently established in the benthic environment (Fernandez et al. 1993a, b; Hunt and Scheibling, 1997; Moksnes et al., 1998; Luppi et al., 2001). In addition, the small size of these individuals may limit their capacity to disperse to habitats with more favorable trophic conditions (Simith et al., 2013).
In our experiment, depending on the specific parameter of juvenile development of L. cumulanta considered (e.g., intermolt period, body size, or % SIM), the adverse effects of nutritional deprivation experienced during the megalopal stage and expressed in the JI crab stage were not necessarily observed in all the other subsequent juvenile stages (JII-JIV or JV). This indicates that the implications of the food restrictions suffered by the megalopae may be reduced or reversed within a few days or weeks at the beginning of juvenile development (Pechenik, 2006). In L. cumulanta, the latent effect of larval starvation on the intermolt period did not persist to subsequent juvenile stages (e.g., JII-JIV). Similarly, juvenile body size (e.g., CW in JIV and CL in JIV and JV crab stages) did not vary significantly between the treatments FC, S1, and S3. However, the preceding larval starvation positively affected the body size of some latter juvenile stages. For example, the largest CW was observed for JII and JIII in S1 and for JV in S3. The same pattern was also observed for the CL of the JII and JIII crab stages when cultured in treatment S1. In the case of growth that was expressed as % SIM, we did not find a clear pattern among the consecutive molt events in response to the different periods of starvation experienced by the megalopae. This reflects a certain degree of intraspecific variability among the subsequent juvenile stages (Hartnoll and Dalley, 1981; Simith et al., 2013). Nevertheless, the most significant increment in size (% SIM) was observed after the first (I-II) and fourth (IV-V) molt events in treatments S1 and S3, respectively, whereas after the third one (III-IV), the juveniles exhibited a smaller size increment in S1 than in the other treatments. We suppose here that the increased or reduced increment in size after these specific molt events may be strongly related to individual variability within the same stage. Furthermore, juveniles produced by larvae originating from only one specific female exhibiting a high or low quality (e.g., nutritional and/or health condition) may have influenced the increase or reduction, respectively, in body size, thus indicating a parental and/or genetic effect on juvenile growth in specific crab stages. In future experiments, decapod larvae from distinct conspecific broods should be cultured individually instead of being mixed, as in the present study. That way the effects of parental condition on larval and juvenile performance would be recorded individually for specimens obtained from distinct conspecific mothers. In the megalopae of Ucides cordatus, the phenotypic effects resulting from the extension of the megalopal stage persisted over a number of juvenile stages and presented an intraspecific variability among juveniles metamorphosed from larvae hatched from different females (Simith et al., 2013).
Our study demonstrates that the timing and duration of the starvation or feeding regime experienced by the megalopae of L. cumulanta may impact their stage duration and metamorphic success. Furthermore, larval starvation may have significant effects (e.g., positive or negative) on the early juvenile biology of this species. These effects varied considerably among the juvenile features (e.g., intermolt period, body size, and growth) and specific crab stages (JI-JV) assessed. This variation observed in the carryover effects of larval food limitation on juvenile crabs could be related to variations in the nutritional condition and/or health of females, or to genetic differences. The reduced survival and the prolonged planktonic period caused by different regimes of larval starvation or feeding may affect the recruitment success, and thus the dynamics and maintenance, of fiddler crab populations within mangrove estuaries.
We thank Suelen Carvalho and Marcus Alexandre Borges Pires for their help during the larval and juvenile culture in the laboratory. We also thank Pro-Reitoria de Pesquisa e Pos-Graduacao (PROPESP) and Fundacao de Amparo e Desenvolvimento da Pesquisa (FADESP) for sponsoring the language translation of the original manuscript through Programa de Apoio a Publicacao Qualificada (PAPQ Program). We are grateful to the editor and two anonymous reviewers for their constructive comments and important corrections on earlier versions of the manuscript. This work was supported by Fundacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) through a doctorate scholarship granted to ASdeS. DJBS received funding from Fundacao Amazonia de Amparo a Estudos e Pesquisas do Estado do Para (FAPESPA) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) through the program for Desenvolvimento Cientifico e Desenvolvimento Tecnologico Regional (DCR; public edict 022/2014; grant 020/2016; processes 2015/145288 and 300549/2016-4). This study is part of the doctorate thesis of ASdeS.
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ADELSON SILVA DE SOUZA (1), TAYSE NASCIMENTO DO ROSARIO (1), DARLAN DE JESUS DE BRITO SIMITH (1,2,9), AND FERNANDO ARAUJO ABRUNHOSA (1)
(1) Laboratorio de Carcinologia, Instituto de Estudos Costeiros (IECOS), Universidade Federal do Para (UFPA), Campus Universitario de Braganca. Alameda Leandro Ribeiro s/n, Aldeia, 68600-000 Braganca, Para, Brasil; and (2) Laboratorio de Ecologia de Manguezal (LAMA), IECOS, UFPA, Campus Universitario de Braganca. Alameda Leandro Ribeiro s/n, Aldeia, 68600-000 Braganga, Para, Brasil
Received 27 February 2018; Accepted 3 December 2018; Published online 19 March 2019.
(*) To whom correspondence should be addressed. Email: firstname.lastname@example.org.
Abbreviations: CL. carapace length; CW, carapace width; F1-F5, feeding treatments 1-5; FC, feeding control; JI-JV, juvenile I-V crab stages; MSD. megalopal stage duration; NVI. Nutritional Vulnerability Index; PNR, point of no return; [PNR.sub.50], period when 50% of the megalopae reach the point of no return; PRS. point of reserve saturation; [PRS.sub.50], period when 50% of the megalopae reach the point of reserve saturation; S1-S5, starvation treatments 1-5; SC, starvation control; % SIM. percentage size increment at molt.
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|Author:||De Souza, Adelson Silva; Do Rosario, Tayse Nascimento; De Jesus De Brito Simith, Darlan; Abrunhosa,|
|Publication:||The Biological Bulletin|
|Date:||Jun 1, 2019|
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