Mar Biol (2007) 152:297–305 DOI 10.1007/s00227-007-0683-3
R ES EA R C H A R TI CLE
Diel cycles of activity, metabolism, and ammonium concentration in tropical holothurians Robert J. Wheeling · E. Alan Verde · James R. Nestler
Received: 15 December 2006 / Accepted: 22 March 2007 / Published online: 13 April 2007 © Springer-Verlag 2007
Abstract Movement rate, oxygen consumption, and respiratory tree ammonium concentration were measured in situ in the holothurians Pearsonothuria graeVei and Holothuria edulis in the Agan-an Marine Reserve, Sibulan, Philippines (9°20⬘30⬙N, 123°18⬘31⬙E). Measurements were made both day and night for both species during April–July 2005. P. graeVei had signiWcantly higher movement rate during the day than at night (1.14 and 0.27 m h¡1, respectively; three-way ANOVA, P < 0.05) while H. edulis had higher movement rate at night compared to the day (0.83 and 0.07 m h¡1, respectively), spending the daylight hours sheltering under coral. More than 80% of H. edulis had movement rate of zero during the day. Oxygen consumption of P. graeVei was signiWcantly higher during the day than at night (1.61 and 0.83 mol O2 g¡1 h¡1, respectively; two-way ANCOVA, P < 0.05), but the reduction at night was not as pronounced as the reduction in movement. H. edulis had a 75% reduction in oxygen consumption during the day compared to night (0.51 and 1.96 mol O2 g¡1 h¡1, respectively), matching this species’ reduced movement rates during the day. Ammonium concentration in water withdrawn from the respiratory trees of P. graeVei during the day (12.0 M) was three times higher than in respiratory tree water sampled at night (4.3 M) and 15 times
Communicated by J.P. Grassle. R. J. Wheeling · J. R. Nestler (&) Department of Biology, Walla Walla University, College Place, WA 99324, USA e-mail:
[email protected] E. A. Verde Corning School of Ocean Studies, Maine Maritime Academy, Castine, ME 04420, USA
higher than ambient seawater (0.8 M; three-way ANOVA, P < 0.05). Ammonium concentration in the respiratory tree water of H. edulis was six times higher at night (14.6 M) than during the day (2.2 M) and 16 times higher than that of ambient seawater (0.9 M). Even though H. edulis and P. graeVei are found within the same coral reef environment, they may aVect diVerent substrates and reef organisms due to their diVerent habitats and distinct but opposite diel cycles.
Introduction Sediment feeding holothurians are signiWcant components of the benthic macrofauna of many tropical and temperate marine communities. These organisms promote bioturbation and recycling of organic material within the sediment (Birkeland 1988; Amon and Herndl 1991; Coulon and Jangoux 1993). Productivity in coral reef environments is higher in the presence of holothurians (Uthicke and Klumpp 1998; Uthicke 2001a), apparently resulting from enhanced nutrient levels (especially nitrogen) in holothurian excretory products (Uthicke 2001b). The eVects of sediment-feeding holothurians on their environment have been the subject of many studies, but how their daily activity rhythms modify these eVects has rarely been demonstrated. In some holothurians activity occurs relatively continuously over a 24 h period (Hamel and Mercier 1998). Most species appear to follow a diel cycle, with maximal activity being diurnal (Shiell 2006), nocturnal (Mercier et al. 1999, 2000), or crepuscular (Graham and Battaglene 2004), depending on the species. These activity cycles can be important for assessing energy and material Xow in food webs, and are triggered by a variety of environmental inputs such as light (Jangoux and Lawrence
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1982), tides (Graham and Battaglene 2004), and food availability (Hamel and Mercier 1998). Two holothurian species, Pearsonothuria graeVei and Holothuria edulis, are found throughout the Indo-PaciWc region (Clark and Rowe 1971), and are in high abundance in certain areas of the central and southern Philippines. These motile species occur in the same reef habitats, occupy a similar depth range (5–20 m), and are approximately the same size (30–40 cm in length). However, P. graeVei occurs mainly on coral and sponge where they appear to graze on epifaunal algal Wlms, while H. edulis is a deposit feeding holothurian that occurs mainly in sandy environments (Samyn 2000; White 2001). These two species may also diVer in their diel cycles. P. graeVei is present throughout the day and night in these locations, while H. edulis is observed only at night (personal observation). The purpose of the present study was to compare the diel cycles of activity (movement rates) and physiological processes (oxygen consumption rates and respiratory tree ammonium concentrations) in these two tropical holothurian species.
Materials and methods Study area and species Research was conducted at the Agan-an Marine Reserve, Sibulan, Philippines (9°20⬘30⬙N, 123°18⬘31⬙E), and the Silliman University Marine Laboratory, Dumaguete, Philippines, during April–July, 2005. Data and samples were collected using SCUBA at depths of 10–20 m. Two species of sea cucumber, Pearsonothuria graeVei (Semper 1868) and Holothuria edulis (Lesson 1830), were used for this study (see Kerr et al. 2005 and Samyn et al. 2005 for current taxonomy of these two genera). Neither species is commercially exploited in this area, and are protected within the Marine Reserve. Data were collected during day (0730–1700 hours) and night (1930–0430 hours). Twilight periods (at least one hour before or after sunset or sunrise) were avoided. Water temperature throughout the study was measured using two HOBO Water Temp Pro loggers located in the central portion of the research area at a depth of 14 m; no signiWcant trends in water temperature occurred during the course of this study, and water temperature did not vary between day and night (28.1 § 0.5°C). Morphological measurements Length (distance from mouth to anus), width (distance from side to side at mid-body), and height (distance from ventral to dorsal surface at mid-body) of undisturbed individuals were measured in situ to the nearest cm with a measuring
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tape for all experiments. The Xexible measuring tape was shaped to follow body curvature. The morphological measurements were used to predict individual wet mass using calibration curves determined for each species. For these curves, 18 P. graeVei and 21 H. edulis of various sizes were placed in an outdoor tank (diameter 3 m, depth 1.5 m) with running seawater for a minimum of 24 h. Length, width, and height were measured for each relaxed and undisturbed individual. Each individual was removed from the tank and handled until water held in the respiratory trees was released through the anus (typically 1–5 min). External water was blotted and individuals were weighed to the nearest g. Individuals were returned to their place of capture within 2 h. Wet mass calibration curves were used to determine in situ mass for 136 P. graeVei (n = 72 for day, n = 64 for night) and 86 H. edulis (n = 39 for day, n = 47 for night). Movement A preliminary experiment was conducted on P. graeVei to determine if tagging with Floy tags (Floy Tag Inc) had an eVect on movement rates. Ten individuals were tagged, and movement rates were measured (see below) after 2 h, 24 h, and two weeks. Tagging and measurements occurred during the day. This preliminary experiment was not conducted on H. edulis as individuals tended to lose tags after less than a week. For subsequent experiments individuals of both species were tagged in situ for identiWcation, and were allowed at least 48 h recovery before data were collected. A series of three dives at 2 h intervals was conducted to monitor in situ movement. During the Wrst dive, Wve to 15 tagged individuals were located. A forestry tape marker with the corresponding tag number was placed 1 cm behind each individual and the time was noted. During each of the subsequent two dives, the linear distance from the posterior end of each individual to its corresponding marker was measured to the nearest cm using a measuring tape, after which the marker was again placed 1 cm behind the individual and the time noted. The mean of the two 2 h distances was determined for each individual. During the study 99 P. graeVei (n = 51 for day, n = 48 for night) and 58 H. edulis (n = 32 for day, n = 26 for night) were used for movement measurements. Metabolism Metabolic rates of 26 untagged P. graeVei (day n = 14, night n = 12) and 21 untagged H. edulis (day n = 11, night n = 10) were measured in situ using an underwater respirometer, allowing for minimal change in water conditions, no exposure to air or temperature Xuctuations during transfers to laboratory conditions, and minimal (if any) changes
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in depth. The respirometer consisted of a clear acrylic chamber (91.4 £ 25.4 £ 25.4 cm) Wtted with a YSI Model 95 oxygen probe and three waterproof battery-operated stirrers. A plastic mesh platform (mesh size 0.5 cm) was placed 5 cm above the bottom of the chamber to keep individuals from directly contacting the oxygen electrode and stirrers. The respirometer was placed at a depth of approximately 10–15 m in the same area as sea cucumbers. Prior to placing an individual in the chamber, oxygen level was measured every 5 min for 30 min to determine background rate of oxygen change. Water in the chamber was Xushed, an individual was placed inside, and oxygen level was measured every 5 min for 2.5 h. Oxygen consumption typically stabilized and remained linear within 15 min; data from the Wrst 30 min of each 2.5 h run were discarded, and only data from the remaining 2 h were used for analysis. Oxygen levels remained above 80% of the starting oxygen value for all individuals. A 0.5 m section of PVC pipe cut in half was placed over H. edulis during the day to mimic this species’ natural covered daytime environment. Individuals were tagged with Floy tags after removal from the respirometer. Oxygen consumption rates were calculated by subtracting the background rate from the holothurian rate of oxygen change, taking into account chamber volume and calculated individual wet mass. Holothurian dry mass and ash-free dry mass may provide a better measure of metabolically active tissue than wet mass (Fraser et al. 2004), but were not determined due to regulations preventing removal or destruction of organisms in the Agan-an Marine Reserve. Ammonium concentration Ammonium concentrations were measured in water removed directly from the respiratory system of untagged cucumbers (P. graeVei n = 19 for day, n = 21 for night; H. edulis n = 12 for day, n = 16 for night). A 10 ml plastic syringe (without needle) was applied to an individual’s anal sphincter during the expiration phase of its respiratory cycle. The sphincter would contract around the end of the syringe, forming a seal, and 5–10 ml of internal water was withdrawn into the syringe. Each individual was tagged with a Floy tag following procurement of the internal water sample. An ambient seawater sample was taken at the same time with a separate syringe approximately 1 m above the cucumber. Syringes were tightly capped for later analysis (within 2 h). Holothurian and ambient seawater samples were double Wltered with 0.45 m syringe Wlters and analyzed for ammonium concentration with a Hitachi Model U-2001 UV/Vis spectrophotometer using the indophenol/ salicylate method (GrasshoV and Kremling 1999). Readings were compared to standard ammonium calibration curves to yield concentrations (M). Ambient seawater
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ammonium concentrations did not signiWcantly diVer between day and night for either species (unpaired t test, P. graeVei t = 1.357, df = 38, P = 0.1827; H. edulis t = 0.699, df = 26, P = 0.4910) and were combined for statistical analyses (Underwood 1981). Statistical analyses Least-squares linear regression analysis was used to predict wet mass from measured and calculated morphological measurements. Data were checked for normality (Kolmogorov–Smirnov test) and homogeneity of variances (Levine’s test), and in the case of nonconformance were Box–Cox transformed (Chen et al. 2002) prior to statistical analysis. Data were back-transformed into original units for presentation. Data from the preliminary experiment to determine the eVect of tagging on movement in P. graeVei were analyzed using repeated measures ANOVA and Tukey HSD multiple comparisons test. Mass, movement rates, and ammonium concentrations were analyzed by three-way mixed-model ANOVA (Bennington and Thayne 1994). Species (P. graeVei and H. edulis) and time period (day and night) were Wxed factors, and month (April–July) was a random factor; water sample (day, night, ambient; Wxed factor) replaced time period in the analysis for ammonium concentrations. Metabolic rates were log transformed and analyzed by ANCOVA with species and time period as the main factors and log body mass as a covariate. Sample sizes for individual months were too low (range 1–4) for each combination of species and time period to include the factor month in the ANCOVA for metabolic rate. If a factor in the ANOVA or ANCOVA analyses was signiWcant, individual means were compared using Tukey HSD post-hoc test. JMP version 6.0.0 (SAS Institute Inc.) was used for all analyses. For all tests the signiWcance level used was 0.05. Data shown are mean § SD.
Results Morphological measurements Calculated elliptical cylinder volume (length £ ½ width £ ½ height £ ) was a better predictor of wet mass for Pearsonothuria graeVei than measured length or calculated rectangular prism volume (length £ width £ height; Table 1), and thus was used to determine mass for this species. Measured length was the best predictor of wet mass for Holothuria edulis (Table 1) and was used for mass determinations for this species. Three-way ANOVA showed that calculated mass did not diVer between day and night for either species (P. graeVei day 792.7 § 184.3 g, n = 72, night 741.0 § 166.5 g, n = 64; H. edulis day
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Table 1 Linear regressions for predicting wet mass of Pearsonothuria graeVei (n = 18) and Holothuria edulis (n = 21) from measured size (length, width, height), volumes calculated from these measured values, and measured wet mass Pearsonothuria graeVei
Holothuria edulis
Regression equation
r
Length vs. mass
M = 24.22 £ L ¡ 148.93
Rectangular prism volume vs. mass
M = 0.44 £ RV + 207.68
Elliptical cylinder volume vs mass
M = 0.56 £ EV + 207.73
2
r2
F
Regression equation
F
0.409
11.06
M = 12.59 £ L ¡ 117.84
0.812
82.21
0.682
47.45
M = 0.36 £ RV + 257.21
0.528
16.77
0.782
57.45
M = 0.34 £ EV + 259.08
0.633
32.70
P for each regression analysis is less than 0.005. L measured length, M measured wet mass, RV calculated rectangular prism volume, EV calculated elliptical cylinder volume
In the preliminary experiment movement rates for P. graeVei were signiWcantly higher two hours after tagging with Floy tags than after 24 h or two weeks (repeated measures ANOVA, F2,18 = 18.19, P < 0.0001, Fig. 1). In the larger
experiment the three-way ANOVA revealed that the interaction between the two species and two time periods was signiWcant (P = 0.001, Table 2). Mean movement rate of P. graeVei was four times higher during the day than at night (Tukey HSD, P < 0.05, Fig. 2). Of the 48 P. graeVei monitored at night 35 had movement rates of zero. Mean movement rate for H. edulis at night was 12 times higher than during the day (Tukey HSD, P < 0.05, Fig. 2). Twenty six of the 32 H. edulis monitored during the day had movement rates of zero.
Table 2 Three-way ANOVA assessing the eVect of species (Pearsonothuria graeVei, Holothuria edulis; Wxed factor), time period (day, night; Wxed factor) or water sample (day, night, ambient; Wxed factor),
and month (April-July; random factor) on the dependent variables body mass, movement rate, and ammonium concentration in tropical holothurians
581.4 § 135.1 g, n = 39, night 567.2 § 150.9, n = 47, P = 0.709), but P. graeVei was signiWcantly larger than H. edulis (P = 0.003, Table 2). Movement
Measurement
Factor
Body mass
Species
1829678.5
P
78.764
0.003
1
40218.7
0.946
0.402
3
50196.3
1.066
0.498
Species £ time period
1
3155.9
0.168
0.709
Species £ month
3
23224.5
1.236
0.433
Time period £ month
3
42647.5
2.270
0.259
3
18786.9
0.782
0.506
196
24037.2
Species
1
0.964
53.853
0.003
Time period
1
0.001
0.006
0.945
Month
3
0.007
0.052
0.984
Species £ time period
1
25.186
145.183
0.001
Species £ month
3
0.017
0.095
0.958
Time period £ month
3
0.110
0.633
0.642
Species £ time period £ month
3
0.174
1.399
0.246
Error
141
0.1244
Species
1
0.1945
Water sample
2
364.953
Month
3
0.303
Species £ water sample
3
117.731
Species £ month
3
0.751
0.738
0.531
Water sample £ month
6
1.071
1.052
0.396
Species £ water sample £ month
6
0.163
0.160
0.987
Error
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1
F
Month
Error
Ammonium concentration
MS
Time period
Species £ time period £ month Movement rate
df
112
1.0173
0.259 341.154 0.189 673.503
0.646
