- Email: [email protected]
Nutritional requirements and starvation resistance in larvae of the brachyuran crabs Sesarma cinereum (Bosc) and S. reticulatum (Say)
J. Exp. Mar. Biol. Ecol., 152 (1991) 271-284 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0022-0981/91/$03.50
271
JEMBE 01664
Nutritional requirements and starvation resistance in larvae of the brachyuran crabs Sesarma cinereum (Bose) and S. reticulatum (Say) Joseph L. Staton ~ and Stephen D. Sulkin2 Department of Biology, University of Southwestern Louisiana, Lafayette, Louisiana, USA" 2 Shannon Point Marine Center, Anacortes, Washington, USA
(Received 19 February 1991; revision received 3 June 1991; accepted 17 June 1991)
Abstract: Nutritional requirements during zoeal development ofSesarma cinereum (Bose) and S. reticulatum (Say) were compared. Both species were raised in the laboratory from hatching to the megalopa on two diets: Artemia nauplii and the rotifer Brachionusplicatilis Muller. Although both species demonstrated a delay in development when fed rotifers, only S. cinereum showed greatly reduced survival. Continuously starved S. cinereum zoeae died during the first zoeal stage, while 17.5% of S. reticulatum larvae successfully molted to Z 2 under starvation conditions. We examined starvation resistance in the Z 1 of S. cinereum and the Z 1 and 3 (terminal zoea) of S. reticulatum by point-of-no-return (PNR) and point-of-reserve saturation (PRS) experiments. The greater starvation resistance in S. reticulatum is correlated with significantly higher lipid content in their larvae as compared to S. cinereum larvae. The high degree of"nutritional flexibility" shown by developing S. reticulatum larvae may be related to other life history traits that characterize its abbreviated larval development. Key words: Crab larva; Nutrition; Sesarma; Starvation resistance
INTRODUCTION
Most brachyuran crabs produce free-living planktotrophic larvae that must feed in the plankton to support completed development. However, brachyuran species differ in their dependence on planktonic feeding for satisfaction of their nutritional requirements via the diet (Sulkin, 1975, 1978; Sulkin & Van Heukelem, 1980; McConaugha, 1982; Levine & Sulkin, 1984a,b). Dietary requirements may vary and depend upon the amount of stored reserves in the egg, the provision of specific kinds of nutrients in the egg, or the ability of larvae to synthesize required nutrients from precursors. Although most brachyuran larvae can withstand periods of food deprivation, the timing and duration of starvation may be critical (Anger & Dawirs, 1981 ; Anger et al., 1981; McConaugha, 1982; Dawirs, 1983; Anger, 1984; Anger & Spindler, 1987). In Correspondence address: J.L. Staton, Department of Biology, Box 42451, University of Southwestern Louisiana, Lafayette, LA 70504-2451, USA. Contribution 2243 from Center for Environmental and Estuarine Studies, University of Maryland, Cambridge, Maryland, USA.
272
J.L. STATON AND S.D. SULKIN
general, early starvation within a zoeal instar is particularly detrimental to successful development, with a point in time beyond which resumption of feeding will not support completed development ("point of no return" w PNR; Anger & Dawirs, 1981). Furthermore, larvae of some species may show a "point of reserve saturation" (PRS) beyond which food intake is not essential to support molting to the next zoeal stage (Anger & Dawirs, 1981). Species also differ in their need to obtain specific nutrients from the diet. For example, larvae of the blue crab Callinectes sapidus Rathbun cannot develop to the megalopa on a diet of rotifers (Brachionus plicatilis Muller cultured on the green alga Dunaliella tertiolecta Butcher), whereas larvae of the deep sea red crab Chaceyon quinquedens (Smith) do as well fed rotifers as they do fed a higher-quality diet ofbrine shrimp (Artemia) nauplii (Sulkin, 1978; Sulkin & Van Heukelem, 1980). Levine & Sulkin (1984a,b) suggest that the former species needs a dietary source of long-chain polyunsaturated fatty acids (PUFA) that are absent in the rotifer, but available in Artemia nauplii. This study examines nutritional requirements in two species of the genus Sesarma. Although Sesarma reticulatum (Say) and S. cinereum (Bose) have similar adult size and inhabit comparable estuarine regions along the east coast of North America, they differ from one another in a variety of reproductive traits that include the number of zoeal instars (Z 1-3 for S. reticulatum and Z 1-4 for S. cinereum) and duration of zoeal development (Costlow & Bookhout, 1960, 1962). We raised larvae of both species on experimental laboratory diets and on starvation treatments designed to compare their response to nutritional stress. Starvation resistance was examined through a comparison of responses of larvae to a range of PRS and PNR treatments (Fig. 1) for the Z 1 of the congeneric crabs S. reticulatum and S. cinereum and between the Z 1 and 3 of S. reticulatum. In addition, the Z 1 of each species was analysed for percent total lipid and fatty acid constituency at hatching.
MATERIALS AND METHODS DIET COMPARISON
We raised groups of larvae of each species to megalopa on diets of the rotifer Brachionus plicatilis (cultured on the alga Dunaiiella) and the nauplii of Artemia. Ovigerous S. cinereum were collected on Pivers Island, near Beaufort, North Carolina, and S. reticulatum, near the mouth of Delaware Bay, and held them individually in 200-mm diameter glass bowls until broods hatched. For S. cinereum, we reared larvae from four broods with each brood represented by 10 bowls (80-mm diameter), each bowl containing 10 larvae. We assigned randomly five of the bowls to each of the two diet treatments for a total of 200 larvae for each diet treatment. Preliminary studies indicated that Z I larvae of S. cinereum were unable to feed effectively on Artemia. The "Artemia" diet treatment, therefore, consisted of rotifers for the Z 1, followed by the brine shrimp nauplii for Z 2 through the last zoeal stage. The "rotifer" diet consisted of rotifers
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
273
throughout zoeal development. For S. reticulatum, we cultured larvae from five broods that were set up as above for a total of 250 larvae for each treatment. In this case, the "Artemia" treatment consisted of nauplii alone from hatching through to the last zoeal stage. For both treatments, we transferred, fed, and recorded life history data for the larvae daily. Starvation resistance Sesanna cinereum.
Ovigerous S. cinereum were held separately in glass bowls, a n d
cultures e x a m i n e d at 3-h intervals. Time of h a t c h i n g w a s set at the m i d p o i n t of the 3-h
interval. PRS experiments consisted of seven treatments shown diagrammatically in Fig. 1. Z 1 differs in duration between the two species (see Results), so increments of feeding/starvation duration were set at 1/6 of average instar duration, respectively. The seven randomized PRS treatments (each with 10 larvae) for S. cinereum thus involved initial feeding for 0, 18, 36, 54, 72, 90 h, and continuous feeding. The PNR experiments consisted of seven randomized treatments with equivalent periods of initial starvation (Fig. 1). Culture water was maintained at 307oo salinity, 25 °C and treated daily prior to use with the antibiotic chloramphenicol (5 rag.l-~) (Fisher & Nelson, 1978). Cultures
PRS
PNR
I II III IV V Vl VII I
II III IV V VI VII Hatching
Molting
Fed
~
Starved
Fig. I. Diagrammatic representation of seven PRS and seven PNR diet treatments showing relative periods of feeding and starvation. Treatments I and VII in both cases involved continuous feeding or starvation from hatching to molting regardless of instar duration. Increments of feeding or starvation in Treatments II-VI were 18 h for S. cinereum and 12 h for S. reticulatum.
274
J.L. STATON AND S.D. SULKIN
were incubated under a light-temperature regime of 12 h : 12 h light: dark and 25 ° C. We assigned larvae from a total of three broods randomly to all treatments for a total of 30 larvae for each of the seven PRS or seven PNR treatments. Sesarma reticulatum Z 1. The same general protocol was followed for Z 1 larvae of S. reticulatum as described above for S. cinereum except that PRS and PNR treatments varied in 12-h increments (e.g., 0, 12, 24, 36, 48, 60 h, and continuously fed or starved control as appropriate; Fig. 1). We assigned larvae from each of two broods to all 14 treatments for a total of 20 larvae for each of the seven PRS or seven PNR treatments. Sesarma reticulatum Z 3. Asynchronous development complicates acquisition of substantial numbers of newly emergent larvae of the target instar, so we utilized the following system to provide sufficient numbers of Z 3 S. reticulatum larvae for the required diet treatments. Upon hatching, we placed individual broods of S. reticulatum larvae into several mass cultures (each < 300 individuals) and fed them either a mixed diet consisting of Artemia nauplii and the rotifer Brachionus plicatilis or only the rotifer. At the start of the Z 3 molt, we collected newly molted larvae in 8-h intervals until a single 8-h collection period produced enough Z 3 zoeae to run an experiment. We assigned randomly Z 3 zoeae among PRS and PNR treatments as described for Z 1 larvae utilizing larvae from three different broods. Two types of experiments were conducted for the Z 3 zoeae; one in which earlier stages of the larvae had been reared on a diet that consisted of a mixture ofrotifers and nauplii and one in which earlier stages were raised on the rotifer alone. DATA ANALYSIS
In the starvation resistance experiments, variances were nonhomogeneous, and we used the nonparametric Dunn's Multiple Comparison test based on Kruskal-Wallis rank sums (Hollander & Wolfe, 1973) to compare time to molt among treatments. In order to evaluate more fully the starvation resistance results, we calculated PRSso and PNRso values from the survival data. For each set of experiments, we plotted survival to each molt against diet treatment and applied linear regression models to the data points defining the maximum slope of the survival curves and thereby estimated the time of feeding or starvation required for 50~o survival (PRSso or PNRso ). LIPID ANALYSIS
Larvae were stored after rinsing in deionized water at - 30 °C pending extraction of lipids from thawed and subsequently vacuum-dried larvae with 2:1 chloroformmethanol solution (Folch et al., 1957). We measured percent lipid of total dry weight gravimetrically, and qualitatively analyzed methylated PUFAs (Morrison & Smith, 1964) using gas chromatography (Supelcowax wide bore capillary column; 60 m; 0.75 mm id).
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
275
RESULTS RESPONSE TO LABORATORY DIETS
S. cinereum
Table I shows percent survival through succeeding zoeal stages for larvae fed both diets. By the end of Z 2, larvae maintained on the rotifer diet showed higher survival than those that had been transferred to Anemia. There was, however, a substantial reduction in survival to the megalopa (survival through Z 4) in larvae fed rotifers compared to Artemia-fed larvae, with greatest mortality occurring during the last zoeal stage among rotifer-fed larvae. Rate of development, as measured by mean day of succeeding molts, is comparable
TABLE I Mean percent survival through succeeding zoeal stages and mean days of succeedirg molts for larvae of S. cinereum and S. reticulatum fed indicated diets. Also shown are results of Student's t tests conducted for each stage (or molt) between diet treatments. Sample sizes (in parentheses) reflect number of bowls used to calculate mean percent survival and number of individual molts used to calculate mean day of molt. Survival values were subject to arcsine transformation for statistical tests. Percent survival Stage S. cinereum
Z Z Z Z
1 2 3 4
Rotifer 99.0 98.5 96.0 61.3
Artemia
(20) (20) (20) (20)
96.8 92.1 91.6 86.3
t test
(19) (19) (19) (19)
NS P < 0.05 NS P < 0.001
4.0 (182) 7.1 (160) 10.8 (166) 15.2 (162)
NS NS P < 0.01 P < 0.001
Mean day of molt Z Z Z Z
1 2 3 4
4.0 (192) 7.3 (188) l l.! (188) 17.3 (121)
Percent survival
S. reticulaturn
Stage
Rotifer
Artemia
Z 1 Z 2 Z 3
98.8 (25) 98.4 (25) 92.6 (25)
98.3 (24) 96.3 (24) 94.8 (24)
t-test ns ns NS
Mean day of molt ZI Z2 Z3
3.6 (235) 6.0 (229) 9.9 (192)
3.3 (225) 5.8 (236) 8.9 (216)
P < 0.001 P < 0.001 P < 0.001
276
J.L. STATON AND S.D. SULKIN
between the two diets through the first two molts (Table I). By Molt III, however, rotifer-fed larvae showed a significant delay in molting, with a similar result seen in mean day to megalopa (Molt IV). In unfed controls, no S. cinereum larvae molted to Z 2, and all unfed larvae died by Day 7 (mean day of death 6.0). S. reticulatum
In contrast to S. cinereum, some unfed S. reticulatum larvae molted successfully to Z 2 (17.5 %). The mean day of Molt I in these unfed larvae was 4.2 + 0.42 (SD). No unfed larva molted successfully to Z 3 and unfed larvae died by Day 11 (mean day of death 8.2). A Student's t test comparing mean days of death between unfed larvae of the two species indicated a significant difference (P < 0.001). S. reticulatum larvae thus survived initial starvation more successfully than did S. cinereum larvae. There was no difference in survival to the megalopa between S. reticulatum larvae fed rotifers and those fed Artemia (Table I). However, S. reticulatum larvae fed rotifers showed a significantly longer (P < 0.001) instar duration as early as the first molt, and at succeeding molts. STARVATION RESISTANCE
S. c#lereum Z 1
In the PNR experiment, extension of the period of initial starvation resulted in reduced survival and prolonged duration of the first zoeal stage in S. cinereum (Table II). Neither continuously starved larvae nor larvae starved for as long as 90 h successfully molted to Z 2. The mean hour to death (mean lifetime in hours for nonsurviving larvae) for the 90-h treatment (180.2 h) was significantly longer than that for the continuously starved treatment (143.4 h; Student's t test, P < 0.001), indicating that provision of food even very late in development did delay mortality. The regression equation was Y = 115.0 - 1.47X for the maximum slope line from the survival data, and the estimate of PNRso was 44.2 h which indicated that 44.2 h of initial starvation resulted in 50~o survival to the succeeding zoeal stage. Given the PNRso = 44.2 h and the mean duration for continuously fed larvae of 112.3 h, we conclude that substantial mortality will occur if initial starvation exceeds ~ 39?/0 of normal instar duration. S. cinereum larvae also were sensitive to starvation imposed after a period of initial feeding (PRS). As the length of the period of initial feeding increased~ survival to Z 2 increased (Table III). No larvae fed for as little as 18 h before continual starvation molted successfully to Z 2. We compared mean hour of death between the 18-h (152.4 h) and starvation treatments (139.2 h) and found that 18 h of initial feeding did delay mortality (Student's t test, P < 0.002) compared with starved controls. The regression equation was Y -- - 75 + 2.778X for the maximum slope line of the survival data and the estimate of PRSso was 45.0 h. Table III lists mean instar duration for larvae surviving to Z 2 ofeach PRS treatment.
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
277
TABLE II Percent survival and mean instar duration (h) ofZ I for each of PNR treatments. For instar duration data, 1 SD and number of molts (n) is presented. * First treatment for which instar duration is significantly different from fed control (Dunn's multiple comparison test, 0c= 0.05). Fed control, 0 initial starvation. Initial starvation (h)
Percent survival
lnstar duration .~
SD
n
S. cinereum
0 18 36 54 72 90 Starved ~
83 60 43 47 10 0 0
112.3 116.0 144.0" 174.9 198.0 -
26.9 21.0 10.6 16.8 -
25 18 13 14 3 0 0
S. reticulatum
0 12 24 36 48 60 Starved ~
100 100 100 100 100 100 45
65.1 74.4 78.6 88.4* 97.3 109.3 96.0
6.2 10.1 7.4 11.6 14.0
19 20 20 19 19 19 9
22.5
18.6
~ Larvae starved continuously from hatching.
TABLE
III
Percent survival and mean instar duration (h) ofZ 1 for each of PRS treatments. 1 SD and number of molts (n) is also presented. * First treatment for which instar duration is significantly different from starved control (Dunn's multiple comparison test, • -- 0.05). Starved control, 0 initial feeding. Initial starvation (h)
S. cinereum
S. reticulatum
0
--
18 36 54 72 90 Fed a
0 17 50 67 63 97
0 12 24 36 48 60 Fed ~
50 60 100 100 100 100 100
a Larvae fed continuously from hatching.
Instar duration
Percent survival
SD
n
--
0
104.4 103.2 107.1 105.2 124.1
8.5 10.9 4.1 12.6 38.6
0 5 15 20 19 29
11.9 9.8 5.6 5.7 5.9 4.9 5.7
10 12 20 20 20 16 20
88.8 78.0 72.0 63.6* 64.2 62.3 68.4
278
J.L. STATON AND S.D. SULKIN
Results from a Kruskal-Wallis ANOVA by ranks indicated no significant differences among the treatments (P > 0.05). Thus extension of the period of initial feeding had no demonstrable effect upon instar duration. Given the PRSso -- 45.0 h and the mean duration of continuously fed larvae of 124.1 h, it appears that mortality will increase substantially if initial feeding is terminated prior to ,~ 36 ~ of normal instar duration. S. reticulatum Z 1
In contrast to the results for S. cinereum, we noted substantial survival to Z 2 in continuously starved S. reticulatum Z I larvae (Tables II, III). In the PNR experiment, the Z 1 survived 100~o to Z 2 in all treatments in which food was presented (Table II). Thus larvae starved for as long as 60 h prior to initiation of feeding survived 100~, and 17.5 ~ of all continuously starved larvae survived. Death in the starved control occurred almost entirely in one of the two broods used in the experiment. Calculation of a meaningful PNRso thus would require narrower treatment intervals than provided in this study. Treatments with increased periods of initial starvation resulted in delayed molting (Table II). The first significant delay in molting occurred at the 36-h treatment. The difference between the 60-h treatment and the starved control was not significant. In the PRS experiment, larvae obtained 100~ survival when initially fed as little as 24 h (Table III). As in the PNR experiments, continuously starved larvae showed substantial survival (50~), with all of the mortality occurring in only one of the two broods used. Calculation of a PRSso is not meaningful under these conditions. An increase in the period of initial feeding resulted in reduction of instar duration (Table III). Results indicated the first significant reduction in instar duration to occur at the 36-h treatment. S. reticulatum Z 3 Z 3 S. reticulatum larvae previously fed the normal mixed diet of brine shrimp nauplii and rotifers could not develop to the megalopa unless food was provided at some point during the instar (Tables IV, V). However, in the PNR experiment, initial starvation imposed for up to 36 h appeared to have little effect upon ultimate survival to the megalopa. Provision of food after 60 h of initial starvation was sufficient to support substantial survival. We generated the regression equation Y = 122.6 - 1.09X from the data and estimated a PNRso of 66.9 h. Although we conducted only one set (unreplicated) of PNR treatments on Z 3 larvae that had previously been fed only on rotifers, obvious differences existed between larvae raised on the two diets. Table V compares the percent survival among PNR treatments for larvae from the same brood previously raised on the two diets. Rotifer-fed larvae were less able to withstand early starvation than were the Artemia-fed group, with 50~o mortality reached after only 24 h of initial starvation.
N U T R I T I O N A L R E Q U I R E M E N T S OF B R A C H Y U R A N LARVAE
279
TABLE IV
S. reticulatum Z 3. Percent survival and mean instar duration (h) for each of PNR treatments. 1 SD and number of molts (n) is also presented. * First treatment for which instar duration is significantly different from fed control (Dunn's multiple comparison test, ~ = 0.05). Fed control, 0 initial starvation. Initial starvation (h)
Percent survival
0 12 24 36 48 60 Starved
Instar duration
93 100 97 90 67 63 0
98.6 110.0 130.3" 138.2 160.8 164.2 -
SD
n
11.6 14.6 25.9 29.0 30.9 45.0 -
28 30 28 27 20 19 0
TABLE V
S. reticulatum Z 3. Percent survival and mean instar duration (h) for each of PNR and PRS treatments in larvae fed either mixed brine shrimp/rotifer diet or only on rotifers prior to and during Z 3. Mixed diet data taken only from same brood as that fed rotifers only. Percent survival
Instar duration
Mixed diet
Rotifer only
Mixed diet
Rotifer only
Initial starved (PNR)
0 12 24 36 48 60 Starved
100 100 100 100 92 75 0
90 90 50 60 30 l0 0
98.0 100.0 113.5 127.0 146.1 138.7 -
117.3 122.7 172.8 160.0 179.7 216.0 -
Initial fed (PRS)
0 12 24 36 48 60 Fed
0 0 58 75 92 100 92
0 0 0 0 78 89 100
113.1 105.3 99.3 97.0 100.4
121.7 108.0 117.3
Mean instar duration was calculated for each diet treatment (larvae previously fed the "mixed" diet) in the PNR experiment for which survival to megalopa occurred (Table IV). Extension of initial starvation prolonged development with the first significant delay occurring at the 24-h treatment (Table IV). Larvae previously fed only rotifers also showed prolonged intermolt period as the period of initial starvation increased (Kruskal-Wallis test, •--0.05). Furthermore, instar duration was significantly longer in rotifer-fed larvae than in those fed the mixed
280
J.L. STATON AND S.D. SULKIN
diet for each of the PNR treatments (Table V; Mann-Whitney U tests for each treatment, • - - 0.05). Larvae fed the mixed diet required initial feeding for at least 24 h prior to starvation before they could survive to megalopa (Table VI). Although initial feeding for only 12 h did not support development to the megalopa, it did result in a delay in death when compared to starved controls (12-h: 194.1 h; starved: 156.8; Student's t test, P < 0.001). Increases in the period ofinitial feeding greater than 24 h increased eventual survival for larvae. We generated the regression equation Y -- - 26.7 + 2.48X from the survival data and calculated a PRSso of 31.0 h or ~ 1/3 of the average duration of continuously fed larvae. Larvae previously fed only on the rotifer required at least 48 h of initial feeding to support survival to the megalopa (Table V). We also calculated mean instar duration for each diet treatment in the PRS experiment that resulted in survival to megalopa (Table VI). Extension of initial feeding period reduced instar duration with significant differences seen only between those fed > 60 h and those fed for 24 h (the minimum time supporting survival to the megalopa; Table VI). TABLE Vl
S. reticulatum Z 3. Percent survival and mean instar duration (h) for each of PRS treatments. 1 SD and number of molts (n) is also presented. * First treatment for which instar duration is significantly different from first treatment that resulted in survival to megalopa (Dunn's multiple comparison test, • = 0.05). Initial feeding (h)
0 12 24 36 48 60 Fed
Instar duration
Percent survival
0 0 33 67 90 90 87
124.8 106.2 102.2 98.2* 99.7
SD
n
43.2 9.9 7.8 4.8 6.7
0 0 10 20 27 27 26
In larvae previously fed only rotifers, only three PRS treatments resulted in survival to the megalopa (Table V). The Kruskal-Wallis test indicated no significant differences among the treatments in instar duration (P > 0.05). In each of the three treatments, instar duration was significantly longer in rotifer-fed larvae than in those fed the mixed diet (Table V, Mann-Whitney U tests, ~ = 0.05).
Lipid analysis We measured lipid content and relative fatty acid content for seven broods of newly hatched larvae for each species. Percent total lipid (+ SE) of S. cinereum was
NUTRITIONAL REQUIREMENTS OF B RACHYURAN LARVAE
281
9.53 + 1.0; S. reticulatum was 12.14 + 0.96. The values were significantly different (Student's t test on arcsine-transformed data, • = 0.05). Analysis of fatty acid constituencies showed highly variable concentrations of 18-C and longer polyunsaturated fatty acids, with variability within species as great as that between species. No differences between species could be distinguished using an average of ordered ranks (Spearman's r, Sokal & Rohlf, 1981).
DISCUSSION
Brachyuran species vary in their ability to develop to the megalopa on an experimental diet of Dunaliella-fed rotifers (S ulkin, 1975; Scott & Middleton, 1979; Levine & Sulkin, 1984b). Levine & Sulkin (1984b) related the inadequacy of the rotifer diet to its low long-chain PUFA content and suggested that differential development among species fed rotifers reflects their differential need to obtain essential fatty acids via fee6ing. Sulkin & Van Heukdem (1980)characterized the ability of some species to develop in the absence of diet high in such PUFA as "nutritional flexibility". Although Sesarma cinereum larvae survive less well when fed rotifers as opposed to Anemia, a large proportion of rotifer-fed larvae do survive to megalopa, and therefore could be categorized as "nutritionally flexible" (Table I). Similar responses are seen in three species of the Xanthidae: Rhithropanopeus harrisii (Gould), Neopanope sayi (Smith) (Sulkin & Norman, 1976), and Eurypanopeus depressus (Smith) (Levine & Sulkin, 1984b). In contrast, S. reticulatum shows a degree of nutritional flexibility that exceeds any reported previously. Not only is survival of zoeae to the megalopa as high on the rotifer diet as on the Artemia diet, but continuously starved larvae are able to survive through the first zoeal molt. Development through the first zoeal instar in the absence of food has been previously reported only in brachyuran species with profoundly abbreviated development (Wear, 1967) or in those that have adapted an essentially terrestrial existence (Sob, 1969; Rabalais & Cameron, 1983, 1985). Resistance to starvation imposed at various intervals during Z I also differed between species. Not surprisingly, provision of food at virtually any point during the first instar resulted in 100 % survival through the first molt in S. reticulatum, although an extended period of feeding early in the instar appeared to produce the best survival (Tables III, IV). The significance of providing food early in the first instar is consistent with results reported for other decapods (Anger, 1987; Anger et al., 1981, 1983, 1985; Anger & Dawirs, 1982; McConaugha, 1982, 1985). In contrast, S. cinereum larvae were more sensitive to extended periods of starvation (Tables III, IV), with results very s" ilar to those reported for S. cinereum by Anger et al. (1981). As in other species, initial starvation extending beyond ,~ 1/3 of the normal zoeal duration results in increased mortality and prolonged instar duration in S. cinereum. Anger (1987) and Anger & Spindler (1987) argue that, if a critical stage in the molt cycle (stage Do; Drach, 1939)
282
J.L. STATON AND S.D. SULKIN
is passed with insufficient nutrient reserves, development may be arrested or death may ensue. Anger (1987) reported that the critical Do threshold occurs from 1/3 to 1/2 of the way through the molting cycle of Z 1 in many decapods. When food was introduced after this PNRso threshold had passed, a few S. cinereum larvae survived to Z 2. The delay in molting in these surviving larvae exceeded the sum of the delay period plus the normal intermolt period which suggests larvae may require replenishment of energy reserves consumed during starvation prior to molting (McConaugha, 1982). Even in those treatments where food was provided too late to support re-initiation of the molt cycle, mortality was still delayed. Even though an essential nutrient for molting has not been identified, a variety of lipids have been implicated. Of particular interest are sterols and other precursors of molting hormones (Anger & Dawirs, 1981; Levine & Sulkin, 1984b; Anger, 1987). Percent total lipid (per dry weight) was 27 % greater in newly hatched S. reticulatum than in S. cinereum. Thus, the relative robustness ofZ 1 S. reticulatum larvae may result from increased lipids (and perhaps essential PUFAs) in the egg which are not found in sufficient concentrations in most other species. Whereas such a difference does not imply causality, this factor does imply a different biochemical composition at hatching which is certainly tied to the species' differential success during starvation. In contrast to the high degree of independence of Z 1 S. reticulatum from food quantity and quality, Z 3 larvae required food at some point during the instar to develop to the megalopa. The PRSso value is the characteristic 1/3 of the normal instar duration, indicating that the instar requires a substantial nutrient reserve to initiate the molt cycle. The PNR experiments illustrated that initiation of feeding could be delayed for as long as 2/3 of the normal third instar duration and still result in substantial survival (Table IV). In those Z 3 larvae that had been cultured on rotifers, resistance to starvation was lower than larvae cultured on a diet including brine shrimp; a longer period of initial feeding was required to promote metamorphosis (Table V) and extended periods of initial starvation resulted in lower survival. Instar duration was dclayed in all treatments. It is apparent that provision of Artemia prior to and during Z 3 resulted in larvae more resistant to food deprivation. Levine & Sulkin (1984a,b) reported that Anemia contain as much as 47% more total lipid per dry weight than do Dunaliella-fed rotifers and are higher in long-chain PUFA, particularly eicosopentaenoic acid (20: 5~o3). We suggest that a diet high in essential lipids early in development provides nutritional flexibility to Z 3 larvae in a manner analogous to that of the egg-stored precursors which promote such flexibility in its Z 1 larvae. Rabalais & Gore (1985) characterized the larval development of S. reticulatum as "advanced" (i.e., hatching stage is more developed), compared to that of S. cinereum. Traits characteristic of this "advanced" life history include a shortened zoeal period, decreased numbers of instars, larger egg sizes, and increased energy reserves at hatching. Thus abbreviated development in many species may be accompanied by an increased incidence of lecithotrophy (Rabalais & Gore, 1985). Such species may pro-
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
283
duce free-living larvae that are "nutritionally flexible". Indeed, in extreme cases, larvae of some species need not feed at all (e.g., Wear, 1967). S. reticulatum demonstrates a high degree of "nutritional flexibility" as well as lecithotrophic tendencies. Such tendencies presumably belong to the complex of traits that characterize abbreviated development in this species. ACKNOWLEDGMENTS
For their assistance on this project, we thank: D.B. Bonar, L.W. Douglass, D.L. Felder, R.B. Griffis, A.H. Hines, V.S. Kennedy, W.B. Van Heukelem, and T.L. Zimmerman for manuscript review; R. Glatter for larval culture; L. A. Franklin, R. I. E. Newell, W.S. Fisher, and T.-J. Chai for chemical analyses; M. Sanborn, J.L. Kyak, and A.R. McGinn for typing, This research was conducted in partial fulfillment of a Masters degree at the University of Maryland (J. L. Staton) and was supported by Maryland Sea Grant 07-5-23082 (S. D. Sulkin), University of Maryland's Horn Point Environmental Laboratories, Western Washington University's Shannon Point Marine Center, and the Department of Biology of the University of Southwestern Louisiana. REFERENCES Anger, K., 1984. Influence of starvation on moult cycle and morphogenesis of Hyas araneus larvae (Decapoda, Majidae). Helgol. Meeresunters., Vol. 38, pp. 21-33. Anger, K., 1987. The Do threshold: a critical point in the larval development of decapod crustaceans. J. Exp. Mar. Biol. Ecol., Vol. 108, pp. 15-30. Anger, K. & R.R. Dawirs, 1981. Influence of starvation on the larval development of Hyas araneus (Decapoda, Majidae). Helgol. Meeresunters., Vol. 34, pp. 287-311. Anger, K. & R. R. Dawirs, 1982. Elemental composition (C,N,H) and energy in growing and starving larvae ofHyas araneus (Decapoda, Majidae). Fish. Bull., Vol. 80, pp. 419-433. Anger, K., R.R. Dawirs, V. Anger & J.D. Costlow, 1981. Effects of early starvation periods on zoeal development of brachyuran crabs. Biol. Bull. (Woods Hole, Mass.), Vol. 161, pp. 199-212. Anger, K., N. Laasch, C. Puschel & F. Schorn, 1983. Changes in biomass and chemical composition of spider crab (Hyas araneus) larvae reared in the laboratory. Mar. Ecol. Prog. Ser., Vol. 12, pp. 91-101. Anger, K. & K.D. Spindler, 1987. Energetics, moult cycle, and ecdysteroid titers in spider crab (Hyas araneus) larvae starved after the D o threshold. Mar. Biol., Vol. 94, pp. 367-375. Anger, K., V. Storch, V. Anger & J. Capuzzo, 1985. Effects of starvation on moult cycle and hepatopancreas of stage I lobster (Homarus americanus) larvae. Helgol. Meeresunters., Vol. 39, pp. 107-116. Costlow, J. D., Jr. & C.G. Bookhout, 1960. The complete larval development of Sesarma cinereum (Bosc) reared in the laboratory. Biol. Bull. (Woods Hole, Mass.), Vol. 118, pp. 203-214. Costlow, J.D., Jr. & e.G. Bookhout, 1962. The larval development of Sesarma reticulatum Say reared in the laboratory. Crustaceana, Vol. 4, pp. 281-294. Dawirs, R.R., 1983. Respiration, energy, balance, and development during growth and starvation of Carcinus maenas L. larvae (Decapoda: Portunidae). J. Exp. Mar. Biol. Ecol., Vol. 69, pp. 105-128. Drach, P., 1939. Mue et cycle d'intermue chez les crustac6s d6capodes. Ann. Inst. Oceanogr. (Monaco), Vol. 19, pp. 103-391. Fisher, W. S. & R.T. Nelson, 1978. Application of antibiotics in the cultivation of Dungeness crab Cancer magister. J. Fish. Res. Board Can., Vol. 35, pp. 1343-1349. Folch, J., M. Lees & G. H. S. Stanley, 1957. A simple method for the isolation and purification of total l;pid from animal tissues. J. Biol. Chem., Vol. 266a, pp. 497-509.
284
J.L. STATON AND S.D. SULKIN
Hollander, M. & D.A. Wolfe, 1973. Non-parametric statistical methods. John Wiley & Sons, New York, 503 pp. Levine, D.M. & S.D. Sulkin, 1984a. Ingestion and assimilation of microencapsulated diets by brachyuran crab larvae. Mar. Biol. Lett., Vol. 5, pp. 147-153. I.evine, D.M. & S.D. Sulkin, 1984b. Nutritional significance of long chain polyunsaturated fatty acids to the zoeal development of the brachyuran crab Eurypanopeus depressus Smith. J. Exp. Mar. BioL Ecol., Vol. 81, pp. 211-223. McConaugha, J. R., 1982. Regulation of crustacean morphogenesis in larvae of the mud crab Rhithropanopeus harrisii. J. Exp. Zooi., Vol. 223, pp. 155-163. McConaugha, J.R., 1985. Nutrition and larval growth. In, Larval growth, edited by A.M. Wenner, A.A. Balkema, Rotterdam, pp. ! 27-154. Morrison, W. R. & L.H. Smith, 1964. Preparation of fatty acid esters and dimethylacetals from lipids with boron-fluoride methanol. J. Lipid Res., Vol. 5, pp. 600-608. Rabalais, N.N. & J.N. Cameron, 1983. Abbreviated development in Uca subcylindrica (Stimpson, 1859) (Crustacea, Decapoda, Ocypodidae) reared in the laboratory. J. Crust. Biol., Vol. 3, pp. 519-541. Rabalais, N, N. & J.N. Cameron, 1985. The effects of factors important in semi-arid environments on the early development of Uca subcylindrica. Biol. Bull. (Woods Hole, Mass.), Vol. 168, pp. 147-160. Rabalais, N.N. & R.H. Gore, 1985. Abbreviated development in decapods. In, Larval growth, edited by A . M . W e n n e r , A . A . Baikema, Rotterdam, pp. 67-126. Scott, A.P. & C. Middleton, 1979. Unicellular algae as food for turbot ($cophthalmus maximus L.) larvae - the: importance of long-chain polyunsaturated fatty acids. Aquaculture, Voi. 14, pp. 247-260. Soh, C.L., 1969. Abbreviated development in a non-marine crab Sesarma (Geosesarma)perraceae (Brachyura; Grapsidae) from Singapore. J. Zool. London, Vol. 158, pp. 357-370. Sokal, R.R. & F.J. Rohlf, 1981. Biometry. W.H. Freeman & Co., San Francisco, second edition, 859 pp. Sulkin, S.D., 1975. The significance of diet in the growth and dex'elopment of larvae of the blue crab, Callinectes sapidus Rathbun, under laboratory conditions. J. Exp. Mar. Biol. Ecol., Vol. 20, pp. 119-135. Sulkin, S.D., 1978. Nutritional requirements during larval development of the portunid crab, Callinectes sapidus Rathbun. J. Exp. Mar. Biol. Ecol., Vol. 34, pp. 29-41. Suikin, S.D. & K. Norman, 1976. A comparison of two diets in the laboratory culture of the zoeal stages of the brachyuran crabs, Rhithropanopeus harrisii and Neopanope sp. Helgol. Wiss. Meeresunters., Vol. 28, pp. 183-190. Sulkin, S. D. & W. F, Van Heukelem, 1980. Ecological and evo~~tionary significance of nutritional flexibility in planktonic larvae of the deep sea red crab Geryon quinquedens and the stone crab Menippe mercenaria. Mar. Ecol. Prog. Set., Vol. 2, pp. 9 !-95. Wear, R.G., 1967. Life history studies of New Zealand Brachyura 1. Embryonic and post-embryonic development of Pilumnus novaezelandiae and P. lumpinus. N.Z. J. Mar. Freshwater Res., Vol. 1, pp. 482-535.
271
JEMBE 01664
Nutritional requirements and starvation resistance in larvae of the brachyuran crabs Sesarma cinereum (Bose) and S. reticulatum (Say) Joseph L. Staton ~ and Stephen D. Sulkin2 Department of Biology, University of Southwestern Louisiana, Lafayette, Louisiana, USA" 2 Shannon Point Marine Center, Anacortes, Washington, USA
(Received 19 February 1991; revision received 3 June 1991; accepted 17 June 1991)
Abstract: Nutritional requirements during zoeal development ofSesarma cinereum (Bose) and S. reticulatum (Say) were compared. Both species were raised in the laboratory from hatching to the megalopa on two diets: Artemia nauplii and the rotifer Brachionusplicatilis Muller. Although both species demonstrated a delay in development when fed rotifers, only S. cinereum showed greatly reduced survival. Continuously starved S. cinereum zoeae died during the first zoeal stage, while 17.5% of S. reticulatum larvae successfully molted to Z 2 under starvation conditions. We examined starvation resistance in the Z 1 of S. cinereum and the Z 1 and 3 (terminal zoea) of S. reticulatum by point-of-no-return (PNR) and point-of-reserve saturation (PRS) experiments. The greater starvation resistance in S. reticulatum is correlated with significantly higher lipid content in their larvae as compared to S. cinereum larvae. The high degree of"nutritional flexibility" shown by developing S. reticulatum larvae may be related to other life history traits that characterize its abbreviated larval development. Key words: Crab larva; Nutrition; Sesarma; Starvation resistance
INTRODUCTION
Most brachyuran crabs produce free-living planktotrophic larvae that must feed in the plankton to support completed development. However, brachyuran species differ in their dependence on planktonic feeding for satisfaction of their nutritional requirements via the diet (Sulkin, 1975, 1978; Sulkin & Van Heukelem, 1980; McConaugha, 1982; Levine & Sulkin, 1984a,b). Dietary requirements may vary and depend upon the amount of stored reserves in the egg, the provision of specific kinds of nutrients in the egg, or the ability of larvae to synthesize required nutrients from precursors. Although most brachyuran larvae can withstand periods of food deprivation, the timing and duration of starvation may be critical (Anger & Dawirs, 1981 ; Anger et al., 1981; McConaugha, 1982; Dawirs, 1983; Anger, 1984; Anger & Spindler, 1987). In Correspondence address: J.L. Staton, Department of Biology, Box 42451, University of Southwestern Louisiana, Lafayette, LA 70504-2451, USA. Contribution 2243 from Center for Environmental and Estuarine Studies, University of Maryland, Cambridge, Maryland, USA.
272
J.L. STATON AND S.D. SULKIN
general, early starvation within a zoeal instar is particularly detrimental to successful development, with a point in time beyond which resumption of feeding will not support completed development ("point of no return" w PNR; Anger & Dawirs, 1981). Furthermore, larvae of some species may show a "point of reserve saturation" (PRS) beyond which food intake is not essential to support molting to the next zoeal stage (Anger & Dawirs, 1981). Species also differ in their need to obtain specific nutrients from the diet. For example, larvae of the blue crab Callinectes sapidus Rathbun cannot develop to the megalopa on a diet of rotifers (Brachionus plicatilis Muller cultured on the green alga Dunaliella tertiolecta Butcher), whereas larvae of the deep sea red crab Chaceyon quinquedens (Smith) do as well fed rotifers as they do fed a higher-quality diet ofbrine shrimp (Artemia) nauplii (Sulkin, 1978; Sulkin & Van Heukelem, 1980). Levine & Sulkin (1984a,b) suggest that the former species needs a dietary source of long-chain polyunsaturated fatty acids (PUFA) that are absent in the rotifer, but available in Artemia nauplii. This study examines nutritional requirements in two species of the genus Sesarma. Although Sesarma reticulatum (Say) and S. cinereum (Bose) have similar adult size and inhabit comparable estuarine regions along the east coast of North America, they differ from one another in a variety of reproductive traits that include the number of zoeal instars (Z 1-3 for S. reticulatum and Z 1-4 for S. cinereum) and duration of zoeal development (Costlow & Bookhout, 1960, 1962). We raised larvae of both species on experimental laboratory diets and on starvation treatments designed to compare their response to nutritional stress. Starvation resistance was examined through a comparison of responses of larvae to a range of PRS and PNR treatments (Fig. 1) for the Z 1 of the congeneric crabs S. reticulatum and S. cinereum and between the Z 1 and 3 of S. reticulatum. In addition, the Z 1 of each species was analysed for percent total lipid and fatty acid constituency at hatching.
MATERIALS AND METHODS DIET COMPARISON
We raised groups of larvae of each species to megalopa on diets of the rotifer Brachionus plicatilis (cultured on the alga Dunaiiella) and the nauplii of Artemia. Ovigerous S. cinereum were collected on Pivers Island, near Beaufort, North Carolina, and S. reticulatum, near the mouth of Delaware Bay, and held them individually in 200-mm diameter glass bowls until broods hatched. For S. cinereum, we reared larvae from four broods with each brood represented by 10 bowls (80-mm diameter), each bowl containing 10 larvae. We assigned randomly five of the bowls to each of the two diet treatments for a total of 200 larvae for each diet treatment. Preliminary studies indicated that Z I larvae of S. cinereum were unable to feed effectively on Artemia. The "Artemia" diet treatment, therefore, consisted of rotifers for the Z 1, followed by the brine shrimp nauplii for Z 2 through the last zoeal stage. The "rotifer" diet consisted of rotifers
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
273
throughout zoeal development. For S. reticulatum, we cultured larvae from five broods that were set up as above for a total of 250 larvae for each treatment. In this case, the "Artemia" treatment consisted of nauplii alone from hatching through to the last zoeal stage. For both treatments, we transferred, fed, and recorded life history data for the larvae daily. Starvation resistance Sesanna cinereum.
Ovigerous S. cinereum were held separately in glass bowls, a n d
cultures e x a m i n e d at 3-h intervals. Time of h a t c h i n g w a s set at the m i d p o i n t of the 3-h
interval. PRS experiments consisted of seven treatments shown diagrammatically in Fig. 1. Z 1 differs in duration between the two species (see Results), so increments of feeding/starvation duration were set at 1/6 of average instar duration, respectively. The seven randomized PRS treatments (each with 10 larvae) for S. cinereum thus involved initial feeding for 0, 18, 36, 54, 72, 90 h, and continuous feeding. The PNR experiments consisted of seven randomized treatments with equivalent periods of initial starvation (Fig. 1). Culture water was maintained at 307oo salinity, 25 °C and treated daily prior to use with the antibiotic chloramphenicol (5 rag.l-~) (Fisher & Nelson, 1978). Cultures
PRS
PNR
I II III IV V Vl VII I
II III IV V VI VII Hatching
Molting
Fed
~
Starved
Fig. I. Diagrammatic representation of seven PRS and seven PNR diet treatments showing relative periods of feeding and starvation. Treatments I and VII in both cases involved continuous feeding or starvation from hatching to molting regardless of instar duration. Increments of feeding or starvation in Treatments II-VI were 18 h for S. cinereum and 12 h for S. reticulatum.
274
J.L. STATON AND S.D. SULKIN
were incubated under a light-temperature regime of 12 h : 12 h light: dark and 25 ° C. We assigned larvae from a total of three broods randomly to all treatments for a total of 30 larvae for each of the seven PRS or seven PNR treatments. Sesarma reticulatum Z 1. The same general protocol was followed for Z 1 larvae of S. reticulatum as described above for S. cinereum except that PRS and PNR treatments varied in 12-h increments (e.g., 0, 12, 24, 36, 48, 60 h, and continuously fed or starved control as appropriate; Fig. 1). We assigned larvae from each of two broods to all 14 treatments for a total of 20 larvae for each of the seven PRS or seven PNR treatments. Sesarma reticulatum Z 3. Asynchronous development complicates acquisition of substantial numbers of newly emergent larvae of the target instar, so we utilized the following system to provide sufficient numbers of Z 3 S. reticulatum larvae for the required diet treatments. Upon hatching, we placed individual broods of S. reticulatum larvae into several mass cultures (each < 300 individuals) and fed them either a mixed diet consisting of Artemia nauplii and the rotifer Brachionus plicatilis or only the rotifer. At the start of the Z 3 molt, we collected newly molted larvae in 8-h intervals until a single 8-h collection period produced enough Z 3 zoeae to run an experiment. We assigned randomly Z 3 zoeae among PRS and PNR treatments as described for Z 1 larvae utilizing larvae from three different broods. Two types of experiments were conducted for the Z 3 zoeae; one in which earlier stages of the larvae had been reared on a diet that consisted of a mixture ofrotifers and nauplii and one in which earlier stages were raised on the rotifer alone. DATA ANALYSIS
In the starvation resistance experiments, variances were nonhomogeneous, and we used the nonparametric Dunn's Multiple Comparison test based on Kruskal-Wallis rank sums (Hollander & Wolfe, 1973) to compare time to molt among treatments. In order to evaluate more fully the starvation resistance results, we calculated PRSso and PNRso values from the survival data. For each set of experiments, we plotted survival to each molt against diet treatment and applied linear regression models to the data points defining the maximum slope of the survival curves and thereby estimated the time of feeding or starvation required for 50~o survival (PRSso or PNRso ). LIPID ANALYSIS
Larvae were stored after rinsing in deionized water at - 30 °C pending extraction of lipids from thawed and subsequently vacuum-dried larvae with 2:1 chloroformmethanol solution (Folch et al., 1957). We measured percent lipid of total dry weight gravimetrically, and qualitatively analyzed methylated PUFAs (Morrison & Smith, 1964) using gas chromatography (Supelcowax wide bore capillary column; 60 m; 0.75 mm id).
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
275
RESULTS RESPONSE TO LABORATORY DIETS
S. cinereum
Table I shows percent survival through succeeding zoeal stages for larvae fed both diets. By the end of Z 2, larvae maintained on the rotifer diet showed higher survival than those that had been transferred to Anemia. There was, however, a substantial reduction in survival to the megalopa (survival through Z 4) in larvae fed rotifers compared to Artemia-fed larvae, with greatest mortality occurring during the last zoeal stage among rotifer-fed larvae. Rate of development, as measured by mean day of succeeding molts, is comparable
TABLE I Mean percent survival through succeeding zoeal stages and mean days of succeedirg molts for larvae of S. cinereum and S. reticulatum fed indicated diets. Also shown are results of Student's t tests conducted for each stage (or molt) between diet treatments. Sample sizes (in parentheses) reflect number of bowls used to calculate mean percent survival and number of individual molts used to calculate mean day of molt. Survival values were subject to arcsine transformation for statistical tests. Percent survival Stage S. cinereum
Z Z Z Z
1 2 3 4
Rotifer 99.0 98.5 96.0 61.3
Artemia
(20) (20) (20) (20)
96.8 92.1 91.6 86.3
t test
(19) (19) (19) (19)
NS P < 0.05 NS P < 0.001
4.0 (182) 7.1 (160) 10.8 (166) 15.2 (162)
NS NS P < 0.01 P < 0.001
Mean day of molt Z Z Z Z
1 2 3 4
4.0 (192) 7.3 (188) l l.! (188) 17.3 (121)
Percent survival
S. reticulaturn
Stage
Rotifer
Artemia
Z 1 Z 2 Z 3
98.8 (25) 98.4 (25) 92.6 (25)
98.3 (24) 96.3 (24) 94.8 (24)
t-test ns ns NS
Mean day of molt ZI Z2 Z3
3.6 (235) 6.0 (229) 9.9 (192)
3.3 (225) 5.8 (236) 8.9 (216)
P < 0.001 P < 0.001 P < 0.001
276
J.L. STATON AND S.D. SULKIN
between the two diets through the first two molts (Table I). By Molt III, however, rotifer-fed larvae showed a significant delay in molting, with a similar result seen in mean day to megalopa (Molt IV). In unfed controls, no S. cinereum larvae molted to Z 2, and all unfed larvae died by Day 7 (mean day of death 6.0). S. reticulatum
In contrast to S. cinereum, some unfed S. reticulatum larvae molted successfully to Z 2 (17.5 %). The mean day of Molt I in these unfed larvae was 4.2 + 0.42 (SD). No unfed larva molted successfully to Z 3 and unfed larvae died by Day 11 (mean day of death 8.2). A Student's t test comparing mean days of death between unfed larvae of the two species indicated a significant difference (P < 0.001). S. reticulatum larvae thus survived initial starvation more successfully than did S. cinereum larvae. There was no difference in survival to the megalopa between S. reticulatum larvae fed rotifers and those fed Artemia (Table I). However, S. reticulatum larvae fed rotifers showed a significantly longer (P < 0.001) instar duration as early as the first molt, and at succeeding molts. STARVATION RESISTANCE
S. c#lereum Z 1
In the PNR experiment, extension of the period of initial starvation resulted in reduced survival and prolonged duration of the first zoeal stage in S. cinereum (Table II). Neither continuously starved larvae nor larvae starved for as long as 90 h successfully molted to Z 2. The mean hour to death (mean lifetime in hours for nonsurviving larvae) for the 90-h treatment (180.2 h) was significantly longer than that for the continuously starved treatment (143.4 h; Student's t test, P < 0.001), indicating that provision of food even very late in development did delay mortality. The regression equation was Y = 115.0 - 1.47X for the maximum slope line from the survival data, and the estimate of PNRso was 44.2 h which indicated that 44.2 h of initial starvation resulted in 50~o survival to the succeeding zoeal stage. Given the PNRso = 44.2 h and the mean duration for continuously fed larvae of 112.3 h, we conclude that substantial mortality will occur if initial starvation exceeds ~ 39?/0 of normal instar duration. S. cinereum larvae also were sensitive to starvation imposed after a period of initial feeding (PRS). As the length of the period of initial feeding increased~ survival to Z 2 increased (Table III). No larvae fed for as little as 18 h before continual starvation molted successfully to Z 2. We compared mean hour of death between the 18-h (152.4 h) and starvation treatments (139.2 h) and found that 18 h of initial feeding did delay mortality (Student's t test, P < 0.002) compared with starved controls. The regression equation was Y -- - 75 + 2.778X for the maximum slope line of the survival data and the estimate of PRSso was 45.0 h. Table III lists mean instar duration for larvae surviving to Z 2 ofeach PRS treatment.
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
277
TABLE II Percent survival and mean instar duration (h) ofZ I for each of PNR treatments. For instar duration data, 1 SD and number of molts (n) is presented. * First treatment for which instar duration is significantly different from fed control (Dunn's multiple comparison test, 0c= 0.05). Fed control, 0 initial starvation. Initial starvation (h)
Percent survival
lnstar duration .~
SD
n
S. cinereum
0 18 36 54 72 90 Starved ~
83 60 43 47 10 0 0
112.3 116.0 144.0" 174.9 198.0 -
26.9 21.0 10.6 16.8 -
25 18 13 14 3 0 0
S. reticulatum
0 12 24 36 48 60 Starved ~
100 100 100 100 100 100 45
65.1 74.4 78.6 88.4* 97.3 109.3 96.0
6.2 10.1 7.4 11.6 14.0
19 20 20 19 19 19 9
22.5
18.6
~ Larvae starved continuously from hatching.
TABLE
III
Percent survival and mean instar duration (h) ofZ 1 for each of PRS treatments. 1 SD and number of molts (n) is also presented. * First treatment for which instar duration is significantly different from starved control (Dunn's multiple comparison test, • -- 0.05). Starved control, 0 initial feeding. Initial starvation (h)
S. cinereum
S. reticulatum
0
--
18 36 54 72 90 Fed a
0 17 50 67 63 97
0 12 24 36 48 60 Fed ~
50 60 100 100 100 100 100
a Larvae fed continuously from hatching.
Instar duration
Percent survival
SD
n
--
0
104.4 103.2 107.1 105.2 124.1
8.5 10.9 4.1 12.6 38.6
0 5 15 20 19 29
11.9 9.8 5.6 5.7 5.9 4.9 5.7
10 12 20 20 20 16 20
88.8 78.0 72.0 63.6* 64.2 62.3 68.4
278
J.L. STATON AND S.D. SULKIN
Results from a Kruskal-Wallis ANOVA by ranks indicated no significant differences among the treatments (P > 0.05). Thus extension of the period of initial feeding had no demonstrable effect upon instar duration. Given the PRSso -- 45.0 h and the mean duration of continuously fed larvae of 124.1 h, it appears that mortality will increase substantially if initial feeding is terminated prior to ,~ 36 ~ of normal instar duration. S. reticulatum Z 1
In contrast to the results for S. cinereum, we noted substantial survival to Z 2 in continuously starved S. reticulatum Z I larvae (Tables II, III). In the PNR experiment, the Z 1 survived 100~o to Z 2 in all treatments in which food was presented (Table II). Thus larvae starved for as long as 60 h prior to initiation of feeding survived 100~, and 17.5 ~ of all continuously starved larvae survived. Death in the starved control occurred almost entirely in one of the two broods used in the experiment. Calculation of a meaningful PNRso thus would require narrower treatment intervals than provided in this study. Treatments with increased periods of initial starvation resulted in delayed molting (Table II). The first significant delay in molting occurred at the 36-h treatment. The difference between the 60-h treatment and the starved control was not significant. In the PRS experiment, larvae obtained 100~ survival when initially fed as little as 24 h (Table III). As in the PNR experiments, continuously starved larvae showed substantial survival (50~), with all of the mortality occurring in only one of the two broods used. Calculation of a PRSso is not meaningful under these conditions. An increase in the period of initial feeding resulted in reduction of instar duration (Table III). Results indicated the first significant reduction in instar duration to occur at the 36-h treatment. S. reticulatum Z 3 Z 3 S. reticulatum larvae previously fed the normal mixed diet of brine shrimp nauplii and rotifers could not develop to the megalopa unless food was provided at some point during the instar (Tables IV, V). However, in the PNR experiment, initial starvation imposed for up to 36 h appeared to have little effect upon ultimate survival to the megalopa. Provision of food after 60 h of initial starvation was sufficient to support substantial survival. We generated the regression equation Y = 122.6 - 1.09X from the data and estimated a PNRso of 66.9 h. Although we conducted only one set (unreplicated) of PNR treatments on Z 3 larvae that had previously been fed only on rotifers, obvious differences existed between larvae raised on the two diets. Table V compares the percent survival among PNR treatments for larvae from the same brood previously raised on the two diets. Rotifer-fed larvae were less able to withstand early starvation than were the Artemia-fed group, with 50~o mortality reached after only 24 h of initial starvation.
N U T R I T I O N A L R E Q U I R E M E N T S OF B R A C H Y U R A N LARVAE
279
TABLE IV
S. reticulatum Z 3. Percent survival and mean instar duration (h) for each of PNR treatments. 1 SD and number of molts (n) is also presented. * First treatment for which instar duration is significantly different from fed control (Dunn's multiple comparison test, ~ = 0.05). Fed control, 0 initial starvation. Initial starvation (h)
Percent survival
0 12 24 36 48 60 Starved
Instar duration
93 100 97 90 67 63 0
98.6 110.0 130.3" 138.2 160.8 164.2 -
SD
n
11.6 14.6 25.9 29.0 30.9 45.0 -
28 30 28 27 20 19 0
TABLE V
S. reticulatum Z 3. Percent survival and mean instar duration (h) for each of PNR and PRS treatments in larvae fed either mixed brine shrimp/rotifer diet or only on rotifers prior to and during Z 3. Mixed diet data taken only from same brood as that fed rotifers only. Percent survival
Instar duration
Mixed diet
Rotifer only
Mixed diet
Rotifer only
Initial starved (PNR)
0 12 24 36 48 60 Starved
100 100 100 100 92 75 0
90 90 50 60 30 l0 0
98.0 100.0 113.5 127.0 146.1 138.7 -
117.3 122.7 172.8 160.0 179.7 216.0 -
Initial fed (PRS)
0 12 24 36 48 60 Fed
0 0 58 75 92 100 92
0 0 0 0 78 89 100
113.1 105.3 99.3 97.0 100.4
121.7 108.0 117.3
Mean instar duration was calculated for each diet treatment (larvae previously fed the "mixed" diet) in the PNR experiment for which survival to megalopa occurred (Table IV). Extension of initial starvation prolonged development with the first significant delay occurring at the 24-h treatment (Table IV). Larvae previously fed only rotifers also showed prolonged intermolt period as the period of initial starvation increased (Kruskal-Wallis test, •--0.05). Furthermore, instar duration was significantly longer in rotifer-fed larvae than in those fed the mixed
280
J.L. STATON AND S.D. SULKIN
diet for each of the PNR treatments (Table V; Mann-Whitney U tests for each treatment, • - - 0.05). Larvae fed the mixed diet required initial feeding for at least 24 h prior to starvation before they could survive to megalopa (Table VI). Although initial feeding for only 12 h did not support development to the megalopa, it did result in a delay in death when compared to starved controls (12-h: 194.1 h; starved: 156.8; Student's t test, P < 0.001). Increases in the period ofinitial feeding greater than 24 h increased eventual survival for larvae. We generated the regression equation Y -- - 26.7 + 2.48X from the survival data and calculated a PRSso of 31.0 h or ~ 1/3 of the average duration of continuously fed larvae. Larvae previously fed only on the rotifer required at least 48 h of initial feeding to support survival to the megalopa (Table V). We also calculated mean instar duration for each diet treatment in the PRS experiment that resulted in survival to megalopa (Table VI). Extension of initial feeding period reduced instar duration with significant differences seen only between those fed > 60 h and those fed for 24 h (the minimum time supporting survival to the megalopa; Table VI). TABLE Vl
S. reticulatum Z 3. Percent survival and mean instar duration (h) for each of PRS treatments. 1 SD and number of molts (n) is also presented. * First treatment for which instar duration is significantly different from first treatment that resulted in survival to megalopa (Dunn's multiple comparison test, • = 0.05). Initial feeding (h)
0 12 24 36 48 60 Fed
Instar duration
Percent survival
0 0 33 67 90 90 87
124.8 106.2 102.2 98.2* 99.7
SD
n
43.2 9.9 7.8 4.8 6.7
0 0 10 20 27 27 26
In larvae previously fed only rotifers, only three PRS treatments resulted in survival to the megalopa (Table V). The Kruskal-Wallis test indicated no significant differences among the treatments in instar duration (P > 0.05). In each of the three treatments, instar duration was significantly longer in rotifer-fed larvae than in those fed the mixed diet (Table V, Mann-Whitney U tests, ~ = 0.05).
Lipid analysis We measured lipid content and relative fatty acid content for seven broods of newly hatched larvae for each species. Percent total lipid (+ SE) of S. cinereum was
NUTRITIONAL REQUIREMENTS OF B RACHYURAN LARVAE
281
9.53 + 1.0; S. reticulatum was 12.14 + 0.96. The values were significantly different (Student's t test on arcsine-transformed data, • = 0.05). Analysis of fatty acid constituencies showed highly variable concentrations of 18-C and longer polyunsaturated fatty acids, with variability within species as great as that between species. No differences between species could be distinguished using an average of ordered ranks (Spearman's r, Sokal & Rohlf, 1981).
DISCUSSION
Brachyuran species vary in their ability to develop to the megalopa on an experimental diet of Dunaliella-fed rotifers (S ulkin, 1975; Scott & Middleton, 1979; Levine & Sulkin, 1984b). Levine & Sulkin (1984b) related the inadequacy of the rotifer diet to its low long-chain PUFA content and suggested that differential development among species fed rotifers reflects their differential need to obtain essential fatty acids via fee6ing. Sulkin & Van Heukdem (1980)characterized the ability of some species to develop in the absence of diet high in such PUFA as "nutritional flexibility". Although Sesarma cinereum larvae survive less well when fed rotifers as opposed to Anemia, a large proportion of rotifer-fed larvae do survive to megalopa, and therefore could be categorized as "nutritionally flexible" (Table I). Similar responses are seen in three species of the Xanthidae: Rhithropanopeus harrisii (Gould), Neopanope sayi (Smith) (Sulkin & Norman, 1976), and Eurypanopeus depressus (Smith) (Levine & Sulkin, 1984b). In contrast, S. reticulatum shows a degree of nutritional flexibility that exceeds any reported previously. Not only is survival of zoeae to the megalopa as high on the rotifer diet as on the Artemia diet, but continuously starved larvae are able to survive through the first zoeal molt. Development through the first zoeal instar in the absence of food has been previously reported only in brachyuran species with profoundly abbreviated development (Wear, 1967) or in those that have adapted an essentially terrestrial existence (Sob, 1969; Rabalais & Cameron, 1983, 1985). Resistance to starvation imposed at various intervals during Z I also differed between species. Not surprisingly, provision of food at virtually any point during the first instar resulted in 100 % survival through the first molt in S. reticulatum, although an extended period of feeding early in the instar appeared to produce the best survival (Tables III, IV). The significance of providing food early in the first instar is consistent with results reported for other decapods (Anger, 1987; Anger et al., 1981, 1983, 1985; Anger & Dawirs, 1982; McConaugha, 1982, 1985). In contrast, S. cinereum larvae were more sensitive to extended periods of starvation (Tables III, IV), with results very s" ilar to those reported for S. cinereum by Anger et al. (1981). As in other species, initial starvation extending beyond ,~ 1/3 of the normal zoeal duration results in increased mortality and prolonged instar duration in S. cinereum. Anger (1987) and Anger & Spindler (1987) argue that, if a critical stage in the molt cycle (stage Do; Drach, 1939)
282
J.L. STATON AND S.D. SULKIN
is passed with insufficient nutrient reserves, development may be arrested or death may ensue. Anger (1987) reported that the critical Do threshold occurs from 1/3 to 1/2 of the way through the molting cycle of Z 1 in many decapods. When food was introduced after this PNRso threshold had passed, a few S. cinereum larvae survived to Z 2. The delay in molting in these surviving larvae exceeded the sum of the delay period plus the normal intermolt period which suggests larvae may require replenishment of energy reserves consumed during starvation prior to molting (McConaugha, 1982). Even in those treatments where food was provided too late to support re-initiation of the molt cycle, mortality was still delayed. Even though an essential nutrient for molting has not been identified, a variety of lipids have been implicated. Of particular interest are sterols and other precursors of molting hormones (Anger & Dawirs, 1981; Levine & Sulkin, 1984b; Anger, 1987). Percent total lipid (per dry weight) was 27 % greater in newly hatched S. reticulatum than in S. cinereum. Thus, the relative robustness ofZ 1 S. reticulatum larvae may result from increased lipids (and perhaps essential PUFAs) in the egg which are not found in sufficient concentrations in most other species. Whereas such a difference does not imply causality, this factor does imply a different biochemical composition at hatching which is certainly tied to the species' differential success during starvation. In contrast to the high degree of independence of Z 1 S. reticulatum from food quantity and quality, Z 3 larvae required food at some point during the instar to develop to the megalopa. The PRSso value is the characteristic 1/3 of the normal instar duration, indicating that the instar requires a substantial nutrient reserve to initiate the molt cycle. The PNR experiments illustrated that initiation of feeding could be delayed for as long as 2/3 of the normal third instar duration and still result in substantial survival (Table IV). In those Z 3 larvae that had been cultured on rotifers, resistance to starvation was lower than larvae cultured on a diet including brine shrimp; a longer period of initial feeding was required to promote metamorphosis (Table V) and extended periods of initial starvation resulted in lower survival. Instar duration was dclayed in all treatments. It is apparent that provision of Artemia prior to and during Z 3 resulted in larvae more resistant to food deprivation. Levine & Sulkin (1984a,b) reported that Anemia contain as much as 47% more total lipid per dry weight than do Dunaliella-fed rotifers and are higher in long-chain PUFA, particularly eicosopentaenoic acid (20: 5~o3). We suggest that a diet high in essential lipids early in development provides nutritional flexibility to Z 3 larvae in a manner analogous to that of the egg-stored precursors which promote such flexibility in its Z 1 larvae. Rabalais & Gore (1985) characterized the larval development of S. reticulatum as "advanced" (i.e., hatching stage is more developed), compared to that of S. cinereum. Traits characteristic of this "advanced" life history include a shortened zoeal period, decreased numbers of instars, larger egg sizes, and increased energy reserves at hatching. Thus abbreviated development in many species may be accompanied by an increased incidence of lecithotrophy (Rabalais & Gore, 1985). Such species may pro-
NUTRITIONAL REQUIREMENTS OF BRACHYURAN LARVAE
283
duce free-living larvae that are "nutritionally flexible". Indeed, in extreme cases, larvae of some species need not feed at all (e.g., Wear, 1967). S. reticulatum demonstrates a high degree of "nutritional flexibility" as well as lecithotrophic tendencies. Such tendencies presumably belong to the complex of traits that characterize abbreviated development in this species. ACKNOWLEDGMENTS
For their assistance on this project, we thank: D.B. Bonar, L.W. Douglass, D.L. Felder, R.B. Griffis, A.H. Hines, V.S. Kennedy, W.B. Van Heukelem, and T.L. Zimmerman for manuscript review; R. Glatter for larval culture; L. A. Franklin, R. I. E. Newell, W.S. Fisher, and T.-J. Chai for chemical analyses; M. Sanborn, J.L. Kyak, and A.R. McGinn for typing, This research was conducted in partial fulfillment of a Masters degree at the University of Maryland (J. L. Staton) and was supported by Maryland Sea Grant 07-5-23082 (S. D. Sulkin), University of Maryland's Horn Point Environmental Laboratories, Western Washington University's Shannon Point Marine Center, and the Department of Biology of the University of Southwestern Louisiana. REFERENCES Anger, K., 1984. Influence of starvation on moult cycle and morphogenesis of Hyas araneus larvae (Decapoda, Majidae). Helgol. Meeresunters., Vol. 38, pp. 21-33. Anger, K., 1987. The Do threshold: a critical point in the larval development of decapod crustaceans. J. Exp. Mar. Biol. Ecol., Vol. 108, pp. 15-30. Anger, K. & R.R. Dawirs, 1981. Influence of starvation on the larval development of Hyas araneus (Decapoda, Majidae). Helgol. Meeresunters., Vol. 34, pp. 287-311. Anger, K. & R. R. Dawirs, 1982. Elemental composition (C,N,H) and energy in growing and starving larvae ofHyas araneus (Decapoda, Majidae). Fish. Bull., Vol. 80, pp. 419-433. Anger, K., R.R. Dawirs, V. Anger & J.D. Costlow, 1981. Effects of early starvation periods on zoeal development of brachyuran crabs. Biol. Bull. (Woods Hole, Mass.), Vol. 161, pp. 199-212. Anger, K., N. Laasch, C. Puschel & F. Schorn, 1983. Changes in biomass and chemical composition of spider crab (Hyas araneus) larvae reared in the laboratory. Mar. Ecol. Prog. Ser., Vol. 12, pp. 91-101. Anger, K. & K.D. Spindler, 1987. Energetics, moult cycle, and ecdysteroid titers in spider crab (Hyas araneus) larvae starved after the D o threshold. Mar. Biol., Vol. 94, pp. 367-375. Anger, K., V. Storch, V. Anger & J. Capuzzo, 1985. Effects of starvation on moult cycle and hepatopancreas of stage I lobster (Homarus americanus) larvae. Helgol. Meeresunters., Vol. 39, pp. 107-116. Costlow, J. D., Jr. & C.G. Bookhout, 1960. The complete larval development of Sesarma cinereum (Bosc) reared in the laboratory. Biol. Bull. (Woods Hole, Mass.), Vol. 118, pp. 203-214. Costlow, J.D., Jr. & e.G. Bookhout, 1962. The larval development of Sesarma reticulatum Say reared in the laboratory. Crustaceana, Vol. 4, pp. 281-294. Dawirs, R.R., 1983. Respiration, energy, balance, and development during growth and starvation of Carcinus maenas L. larvae (Decapoda: Portunidae). J. Exp. Mar. Biol. Ecol., Vol. 69, pp. 105-128. Drach, P., 1939. Mue et cycle d'intermue chez les crustac6s d6capodes. Ann. Inst. Oceanogr. (Monaco), Vol. 19, pp. 103-391. Fisher, W. S. & R.T. Nelson, 1978. Application of antibiotics in the cultivation of Dungeness crab Cancer magister. J. Fish. Res. Board Can., Vol. 35, pp. 1343-1349. Folch, J., M. Lees & G. H. S. Stanley, 1957. A simple method for the isolation and purification of total l;pid from animal tissues. J. Biol. Chem., Vol. 266a, pp. 497-509.
284
J.L. STATON AND S.D. SULKIN
Hollander, M. & D.A. Wolfe, 1973. Non-parametric statistical methods. John Wiley & Sons, New York, 503 pp. Levine, D.M. & S.D. Sulkin, 1984a. Ingestion and assimilation of microencapsulated diets by brachyuran crab larvae. Mar. Biol. Lett., Vol. 5, pp. 147-153. I.evine, D.M. & S.D. Sulkin, 1984b. Nutritional significance of long chain polyunsaturated fatty acids to the zoeal development of the brachyuran crab Eurypanopeus depressus Smith. J. Exp. Mar. BioL Ecol., Vol. 81, pp. 211-223. McConaugha, J. R., 1982. Regulation of crustacean morphogenesis in larvae of the mud crab Rhithropanopeus harrisii. J. Exp. Zooi., Vol. 223, pp. 155-163. McConaugha, J.R., 1985. Nutrition and larval growth. In, Larval growth, edited by A.M. Wenner, A.A. Balkema, Rotterdam, pp. ! 27-154. Morrison, W. R. & L.H. Smith, 1964. Preparation of fatty acid esters and dimethylacetals from lipids with boron-fluoride methanol. J. Lipid Res., Vol. 5, pp. 600-608. Rabalais, N.N. & J.N. Cameron, 1983. Abbreviated development in Uca subcylindrica (Stimpson, 1859) (Crustacea, Decapoda, Ocypodidae) reared in the laboratory. J. Crust. Biol., Vol. 3, pp. 519-541. Rabalais, N, N. & J.N. Cameron, 1985. The effects of factors important in semi-arid environments on the early development of Uca subcylindrica. Biol. Bull. (Woods Hole, Mass.), Vol. 168, pp. 147-160. Rabalais, N.N. & R.H. Gore, 1985. Abbreviated development in decapods. In, Larval growth, edited by A . M . W e n n e r , A . A . Baikema, Rotterdam, pp. 67-126. Scott, A.P. & C. Middleton, 1979. Unicellular algae as food for turbot ($cophthalmus maximus L.) larvae - the: importance of long-chain polyunsaturated fatty acids. Aquaculture, Voi. 14, pp. 247-260. Soh, C.L., 1969. Abbreviated development in a non-marine crab Sesarma (Geosesarma)perraceae (Brachyura; Grapsidae) from Singapore. J. Zool. London, Vol. 158, pp. 357-370. Sokal, R.R. & F.J. Rohlf, 1981. Biometry. W.H. Freeman & Co., San Francisco, second edition, 859 pp. Sulkin, S.D., 1975. The significance of diet in the growth and dex'elopment of larvae of the blue crab, Callinectes sapidus Rathbun, under laboratory conditions. J. Exp. Mar. Biol. Ecol., Vol. 20, pp. 119-135. Sulkin, S.D., 1978. Nutritional requirements during larval development of the portunid crab, Callinectes sapidus Rathbun. J. Exp. Mar. Biol. Ecol., Vol. 34, pp. 29-41. Suikin, S.D. & K. Norman, 1976. A comparison of two diets in the laboratory culture of the zoeal stages of the brachyuran crabs, Rhithropanopeus harrisii and Neopanope sp. Helgol. Wiss. Meeresunters., Vol. 28, pp. 183-190. Sulkin, S. D. & W. F, Van Heukelem, 1980. Ecological and evo~~tionary significance of nutritional flexibility in planktonic larvae of the deep sea red crab Geryon quinquedens and the stone crab Menippe mercenaria. Mar. Ecol. Prog. Set., Vol. 2, pp. 9 !-95. Wear, R.G., 1967. Life history studies of New Zealand Brachyura 1. Embryonic and post-embryonic development of Pilumnus novaezelandiae and P. lumpinus. N.Z. J. Mar. Freshwater Res., Vol. 1, pp. 482-535.