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Simultaneous measurements of oxygen consumption and ammonia-N excretion in embryos and larvae of marine invertebrates
Comparative Biochemistry and Physiology Part A 136 (2003) 321–328
Simultaneous measurements of oxygen consumption and ammonia-N excretion in embryos and larvae of marine invertebrates D. Lemosa,*, R.L.V. Jorgeb, V.N. Phana a
b
´ ˜ Paulo, C.P. P.O. Box 66149, Sao ˜ Paulo, SP 05389-900, Brazil Instituto Oceanografico, University of Sao ˜ Carlos, University of Sao ˜ Paulo, C.P. P.O. Box 292, Sao ˜ Carlos, SP 13560-970, Brazil Escola de Engenharia de Sao Received 18 November 2002; received in revised form 2 June 2003; accepted 2 June 2003
Abstract The quantification of oxygen consumption and ammonia-N excretion rates is essential in determining energy requirements for development of larval invertebrates. In larval energetics, there is a need for accurate and uncomplicated techniques to quantify metabolic rates. A method for simultaneous measurements of oxygen and ammonia-N concentrations is presented. It employs sealed respirometric chambers (ca. 30 ml) in which embryos and larvae are incubated. Analysis is carried out in end-point samples by Winkler’s titration and indophenol-blue for oxygen and ammonia-N, respectively. Water is sampled into volume-calibrated glass syringes and oxygen consumption and ammoniaN excretion rates were determined by the difference between experimental and control (no animals) units. The method was successfully used to measure metabolic rates in embryo and larval stages of the shrimp Farfantepenaeus paulensis and in veliger of the mussel Perna perna. The accuracy denoted by the coefficient of variation is comparable to previous results on larval metabolic rates. A biomass: volume (mg mly1 ) is proposed to extend its application to further species of marine invertebrates. The method is simple to operate, involves non-expensive material and is portable enough for field work. A substantial number of replicates can be analyzed at the same time and O:N ratio, an indicator of the catabolized substrate, can be calculated. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Embryo; Excretion; F. paulensis; Larvae; Mussel; Oxygen-consumption; P. perna; Shrimp
1. Introduction Energy requirements for embryonic and larval development of marine invertebrates can be determined based on correct measurements of oxygen consumption and ammonia-N excretion rates. In hatcheries, early ontogenetic stages are often maintained in high densities and data on oxygen demand and ammonia-N release rates are necessary for a correct dimensioning of structures and routine of cultivation. Several methods have been devel*Corresponding author. Fax: q55-11-3091-6607. E-mail address: [email protected] (D. Lemos).
oped for the quantification of oxygen consumption in embryos and larvae of invertebrates such as micro-Cartesian divers (Zeuthen, 1943; Scholander et al., 1952), Winkler’s titration (Bhatnagar and Crisp, 1965) and polarographic oxygen sensors (Gnaiger, 1983; Hoegh-Guldberg and Emlet, 1997) adapted to micro-respiration chambers. Some of them are rather complicated and difficult to handle in field conditions, being not routinely used (Marsh and Manahan, 1999). Few methods have been described for simultaneous determination of oxygen consumption and ammonia-N that enables the calculation of O:N, an indicator of the catab-
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1095-6433(03)00163-6
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olized substrate (Mayzaud and Conover, 1988). The accuracy of the technique is possibly the most important aspect to consider. For instance, continuous measurements of oxygen consumption by polarographic oxygen sensors may underestimate respiration rates compared to Winkler’s titration (Shilling et al., 1996; Marsh and Manahan, 1999). The development of alternative and accurate techniques for determination of larval metabolic rates is thus highly desirable. A method for determination of oxygen consumption and ammonia-N excretion rates in embryos and larval invertebrates is here described. Evaluation of oxygen and ammonia-N were carried out in sealed respirometers through analysis of end-point samples. The technique combines the use of simple equipment with the precision of Winkler’s and indophenol-blue (Koroleff, 1983) procedures for determination of oxygen and ammonia, respectively. It also allows a substantial number of replicates at the same time. The detailed protocol is for first time introduced and efficacy is demonstrated by data on metabolic rates of larval shrimp F. paulensis (Lemos and Phan, 2001a) and mussel P. perna. 2. Material and methods 2.1. Experimental animals Adult shrimps F. paulensis were obtained from the commercial fishery at Santa Catarina State coast, Brazil (278189 Lat S, 488239 Long W). After acclimation to culture conditions, females (33.9"4.7 g wet mass) were induced to sexual maturity by unilateral eyestalk ablation (Wyban et al., 1987), and individually isolated in 500-l fiberglass tanks. Spawning occurred at night, and viable eggs (ca. 200 000 per female) were stocked in cylindrical-conical tanks, hatching 12 to 14 h later at 26"1 8C. Larval development of F. paulensis presents six naupliar (N I to VI), three protozoeal (PZ I to III), and three mysid (M I to III) stages (Iwai, 1978). At nauplius V-VI, larvae were moved to 50 000 l tanks, and reared according to commercial practices at 26"1 8C and 34"1‰ S (Vinatea et al., 1993). Larvae were initially stocked at 100 ind ly1 with strong aeration and a 40–60% water renewal daily. Exogenous food was supplied from the stage PZ I and was made of the diatom Chaetoceros calcitrans (80 000 cells mly1) and artificial plankton (Nippai Shrimp Feed Inc.,
Japan; 0.03 mg larvay1 dayy1, 30 mm particlesize). From PZ III, freshly hatched nauplii of Artemia sp. (5 ind larvay1 dayy1) were added to the diet. The Artemia sp. ration was set to 15 nauplii larvay1 dayy1 between M I and M III. After metamorphosis, postlarvae (PL) were fed microalgae with increasing amounts of artificial plankton and brine shrimp nauplii. Metabolic rates of larval shrimp were determined at 34"1‰ and 26"0.5 8C. Samples of pooled individuals at same developmental stage were selected from a tank containing nine synchronized spawns. A homogeneous sample was characterized when )80% of individuals belonged to the same stage, the remaining individuals differing by only a single stage. Embryos were sampled at the ‘early nauplius’ embryonic stage, 10 to 12 h after spawning (Primavera and Posadas, 1981). Oxygen consumption and ammonia-N excretion were then determined in embryo, N III, PZ I and in each subsequent larval stage until metamorphosis. The first postlarval instar was verified after the molt of metamorphosis, sorted by the number of rostral teeth combined to 6th segment setae (Iwai, 1978). Individuals from different stages were chosen near to the intermolt period that comprises 7–30% of the total molt cycle since the last ecdysis (Dall et al., 1990). ˜ Sebastiao ˜ Mussels P. perna were caught at Sao Island rocky shores (238549 Lat S, 458279 Long W). Spawning was obtained after keeping separated males and females exposed to air at 17.0"2.0 8C for 15 h followed by immersion in UV sterilized seawater at 28.5"2.5 8C and 34"1‰. Sperm and oocytes were separately washed through a sequence of 100 to 22 mm mesh size nets in order to clear from debris. Oocytes were fertilized by the addition of fresh sperm followed by gently shaking and incubation at 24"0.5 8C with smooth aeration. After 27 h, unfed veliger individuals (82.05"6.67 mm) were separated through 100 to 50 mm nets and counted in Sedgewick–Rafter chambers under optical microscope. Experiments were carried out in 34"1‰ and 24"0.5 8C. Larval stages were identified according to Rojas and Martinez (1967). Replicates of pooled shrimp or mussel larvae were rinsed with distilled water, dried with filter paper and separated to mass determination. Shrimp samples were dried for 48 h at 70 8C, and weighed on a Cahn C-31 microbalance to the nearest 0.1
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required for animals to acclimate in the chamber was previously established by measures of oxygen consumption of shrimp protozoea since the beginning of the confinement. After the acclimation period, the net-cover was removed and chambers were carefully closed as the excess water escaped through the cover orifice, expelling the remaining air in between water and cover surface. The plastic tablet was laid over the external excess water covering the orifice and generating a tension to avoid water contact with air. Five to ten replicate experimental chambers were accompanied by 5 controls (no animals) in order to detect any possible oxygen variation due to biological or chemical demand. Units were then placed in a temperature controlled water bath during the incubation. Initial oxygen concentration was near to saturation at that temperature and salinity conditions (Grasshoff, 1976). The time of incubation varied among developmental stages and oxygen levels were not allowed to reach less than 80% saturation (Dawirs, 1983). Fig. 1. Respirometric chamber used in measurements of oxygen consumption ammonia-N excretion of embryos and larvae of marine invertebrates.
mg. After 48 h at 80 8C, mussel larvae were weighed in a Mettler Toledo balance (precision 0.1 mg). 2.2. Respirometric chambers Individuals were placed in cylindrical plastic chambers of ca. 30 ml with a plastic cover that provide hermetical sealing (Fig. 1). An orifice of 1.5 mm placed in the center of the cover allowed the elimination of air bubbles from inside the chamber prior to closing. A plastic tablet covered the orifice isolating inside water by holding a tension between the tablet and the water in the orifice. In our experiments, 35 mm translucent photographic film containers were used showing inert, adequate for handling, ease to obtain and maintain constant volume. Chambers were individually identified and exact volume was determined gravimetrically. A known number of individuals were put into the chambers and filled with clear water to the top of the container. Respirometric units (identified containers) were covered by an adapted plastic cover with a net of adequate mesh size to avoid animal escape, and submitted to flow through clear seawater for acclimation. The time
2.3. Water sampling and analysis Water was sampled by using 10 ml glasssyringes (Becton and Dickinson) equipped with plastic nozzles. Samples contained 8 ml of water in the barrel plus the volume in the dead space in the nozzle. The volume of the dead space in the nozzle was chemically determined in each syringe by filling it with 0.025 N potassium iodate following the addition of 1% (wyv) potassium iodide and the titration against 0.01 N sodium thiosulfate (Fox and Wingfield, 1938). Volume calibrated syringes were used in sampling for oxygen determination. A plastic cannula attached to the tip of the nozzle was introduced into the respirometric chamber by the cover orifice. Two milliliters were drawn up into the barrel and washed out to eliminate bubbles from inside barrel and nozzle. Samples were taken carefully until the mark of 8 ml. Reagents for oxygen determination by the Winkler method wmanganic (0.4 ml) and alkaline (0.8 ml) solutions, phosphoric acid (0.8 ml)x were drawn up directly into the syringes up to the mark of 10 ml. The plunger was locked after covering the nozzle with a plastic nozzle-shaped jacket. Samples were titrated against 0.01 N sodium thiosulfate by using a portable micrometer burette (Gilson Instruments) to the nearest 0.001 ml. The oxygen concentration in samples was calculated
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Table 1 Salt effect factor (SF) to correct ammonia-N values in seawater (after Koroleff, 1983) Salinity (‰) 0 SF
5
10
15
20
25
30
in oxygen or ammonia-N contents. Oxygen consumption and ammonia-N excretion rates were determined by the difference between experimental and control units, and were expressed as individual (mg indy1 hy1) and dry mass-specific (mg gy1 DW hy1) rates. Atomic O:N ratios could be calculated by dividing values of oxygen consumed by the nitrogen excreted in each developmental stage (Mayzaud and Conover, 1988).
35
1.00 1.03 1.06 1.09 1.14 1.18 1.22 1.25
as: wO2x (mg ly1)s8000=N=HyV or wO2x (ml ly1)s5600=N=HyV
2.4. Statistical analysis Differences among means were detected by one way ANOVA followed by Tukey’s multicomparison test. When data did not follow normal distribution, differences were verified by the non-parametric Kruskal–Wallis analysis followed by mean comparison of Nemenyi. Results were considered significant at P-0.05 (Zar, 1984).
where Nsvolume of sodium thiosulfate expended; Hsnormality of sodium thiosulfate; Vssample volume (barrelqnozzle). The solution of sodium thiosulfate used in titration was standardized at regular intervals to determine its exact normality. After sampling for oxygen content, 10 ml were taken from respirometric chambers by glass syringes and put in amber flasks for determination of ammonia-N content. Washing samples (2 ml) were drawn up as for oxygen determination to eliminate bubbles from inside barrel. Solutions of phenoldisodium nitroprusside (R1) and trisodium citratesodium hypochlorite (R2) were added for the formation of indophenol blue (Koroleff, 1983). Reagents were standardized with solution of ammonia chloride dissolved in ammonia-free purified water (Milli Q娃 –Millipore). When analyzing seawater, dissolved salts reduce the indophenol blue produced so that values were corrected by the salt effect factor (Table 1). The eventual presence of organisms drawn up through water samples did not result in difference
3. Results The number of shrimp larvae incubated in respirometric chambers varied according to individual dry mass (Table 2). Five-hundred shrimp embryos were necessary to produce changes in oxygen and ammonia-N concentration while the same was reached by 8 to 130 individuals in the following larval stages. Metabolic rate of nauplius III (N III) with reduced mass compared to embryo was detected in groups of 130 individuals. In the following stages, the number of individuals pooled decreased with the increment in dry mass. Incubation time for each stage varied between 90 (protozoea I and II) and 165 min (N III) and was not related to the number of individuals in chamber. For mussel, a higher number of veliger (4500
Table 2 Age, dry mass (DW), number of individuals, incubation time and biomass: volume ratio in closed respirometers for determination of oxygen consumption and ammonia-N excretion during the early life stages of F. paulensis at 26"0.5 8C. Nsnauplius, PZsprotozoea, Msmysis and PLspostlarva. Roman numerals denote the number of molts in stage Stage
Age (days after spawning)
DW (mg)
Individuals in chamber
Incubation time (min)
Biomass: volume (mg mly1)
Embryo N III PZ I PZ II PZ III MI M II M III PL I
0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5
4.1 2.8 6.6 13.7 24.0 53.5 81.3 91.7 108.7
500 130 80 50 20 15 10 10 8
120 165 90 90 120 150 130 140 120
68.3 12.1 17.6 22.8 16.0 26.7 27.1 30.6 29.0
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Table 3 Age, dry weight (DW), number of individuals, incubation time and biomass: volume ration in closed respirometers for determination of oxygen consumption and ammonia-N excretion in veliger of P. perna at 24"0.5 8C Stage
Age (days after spawning)
DW (mg)
Individuals in chamber
Incubation time (min)
Biomass: volume (mg mly1)
Veliger
1.12
0.0984
4500
300
14.8
further determinations of metabolic rates in other larval species. Acclimation to respirometers was determined by oxygen consumption rates since the moment shrimp larva was placed in the chamber (Fig. 2). Individual oxygen consumption (VO2) of PZ III decreased significantly after 20 min incubation (P-0.05) and stabilized after 100 min in PZ III. In the following experiments, animals were kept for a minimum of 100 min in flow-through seawater prior to close respirometers. The time of incubation required to 80% oxygen saturation varied among shrimp larval stages, extending from ca. 190 min in embryo, N III and PZ II (P)0.05) to 215 min in M I and 310 min in PL I. Individual rates of oxygen consumption (VO2) increased significantly throughout shrimp development (P-0.05) (Table 4). In contrast, massspecific rates (QO2) varied significantly throughout ontogenetic stages. Higher rates were observed in stages N III and PZ I, 30.0 and 24.0 mg O2 gy1 DW hy1, respectively. Mussel veliger presented reduced VO2 and individual ammonia-N excretion compared to shrimp stages (Table 5). However, in mass-specific basis, veliger showed QO2 as high as shrimp protozoeal stages. Individual ammonia-N excretion increased through the majority of shrimp developmental stages, excepting the intervals M I-M II and M III-PL I that presented reduced rates (Table 4).
Fig. 2. Individual oxygen consumption related to time of acclimation to sealed repirometers in protozoea III (PZ III) of F. paulensis at 26"0.5 8C. Results expressed as means, error barssS.D. Different letters denote significant differences (P) 0.05). (Data from Lemos and Phan, 2001a).
ind) was necessary for the determination of metabolic rate as a longer incubation time (Table 3). Though experiments were carried in different temperatures, the biomass-volume ratio (by dividing the total dry mass of individuals by the chamber volume—mg mly1) was 68.3 for benthic embryo and ranged from 12.1 to 30.6 for planktonic larval shrimp and mussel (Tables 2 and 3). At the same temperature, early larval stages required lower biomass-ratios. The biomass–volume ratio can contribute to establish the stocking density in
Table 4 Individual and mass-specific rates of oxygen consumption and ammonia-N excretion, and O:N ratios during the early life stages of the shrimp F. paulensis at 26"0.5 8C. Results expressed as means (S.D. in parentheses). (Data from Lemos and Phan, 2001a) Stage
mg O2 indy1 hy1
Embryo N III PZ I PZ II PZ III MI M II M III PL I
0.012 0.083 0.158 0.212 0.362 0.667 0.859 0.986 1.209
(0.002) (0.015) (0.007) (0.029) (0.108) (0.170) (0.089) (0.098) (0.322)
mg N-NH3 indy1 hy1 0.0012 0.0019 0.0048 0.0103 0.0126 0.0182 0.0112 0.0193 0.0096
(0.00041) (0.00029) (0.00031) (0.0047) (0.0029) (0.0026) (0.0037) (0.0051) (0.0013)
mg O2 g DWy1 hy1 7.6 30.0 24.0 15.5 15.1 12.5 10.6 10.7 11.1
(1.28) (5.40) (1.10) (2.10) (4.49) (3.18) (1.10) (1.07) (2.97)
mg N-NH3 g DWy1 hy1 0.30 0.68 0.73 1.02 0.52 0.34 0.14 0.21 0.09
(0.10) (0.11) (0.05) (0.08) (0.12) (0.05) (0.05) (0.05) (0.01)
O:N 6.5 27.5 19.4 9.0 18.7 17.9 57.4 66.6 82.4
(2.9) (7.3) (1.7) (1.1) (3.1) (2.6) (6.5) (10.5) (8.4)
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Table 5 Individual and mass-specific rates of oxygen consumption and ammonia-N excretion, and O:N ratio of larval mussel P. perna at 24"0.5 8C. Results expressed as means (S.D. in parentheses) Stage
ng O2 indy1 hy1
ng N-NH3 indy1 hy1
mg O2 g DWy1 hy1
mg N-NH3 g DWy1 hy1
O:N
Veliger
1.96 (0.62)
0.443 (0.081)
19.87 (6.22)
4.50 (0.83)
3.7 (1.2)
Mass-specific rates of ammonia-N excretion also varied significantly during development (P0.05). Individual excretion rates were reduced in larval mussel compared to shrimp while massspecific rates were much higher (Table 5). As a general trend, shrimp development was characterized by low O:N values between PZ I and M I, and high values between M II and PL I. Mussel veliger showed decreased O:N value compared to shrimp larvae (Table 5). Data on shrimp oxygen consumption, ammonia-N excretion and O:N performed a mean coefficient of variation (CV) of 17, 23 and 17%, respectively. Variation on rates of larval mussel showed a CV of 31, 18 and 32% for oxygen consumption, ammonia-N excretion and O:N, respectively. 4. Discussion Oxygen consumption is a major energy expense throughout larval development of marine invertebrates and requires precise measurement. However, due to the reduced size of individuals and the small amount of oxygen consumed, sensitive and accurate techniques are necessary. The present method enables simultaneous measurements of oxygen consumption and ammonia-N excretion of invertebrate embryos and larvae by using simple and inexpensive material, with reduced dimensions that can be easily transported. The experimental routine is rather uncomplicated and permits application after a few hours training. It also allows a substantial number of replicates at the same time and slight variations in oxygen and ammonia-N concentrations can be detected. Incubation period mostly depends on metabolic rates and stocking density of individuals. Planktonic larvae must be arranged as to enable freeswimming in the chamber and densities can be calculated from biomass-volume ratios (Tables 2 and 3). At the same temperature, early stages require lower biomass-ratios compared to advanced larval stages. The number of larval shrimp or mussel stocked may eventually be altered from that presented depending on experi-
mental purpose and culture conditions, (e.g. temperature gradient, exposure to pollutants). Experiments with benthic embryos that remain deposited over the bottom during incubation may produce vertical stratification of oxygen and ammonia-N concentration. This concern can be minimized through the collection of a large sample volume as it has been taken for oxygen (ca. 8 ml) and ammonia-N (10 ml) regarding total chamber capacity. Acclimation to respirometric chambers has been reported for juvenile and adult marine animals by decrease in activity and oxygen consumption (Jones and Randall, 1978). Accordingly, animals previously subjected to stresses of handling and space limitation adapt to experimental chambers after being submitted to certain conditions as clear and oxygenated water, absence of external movement and visual disturbance (Buttle et al., 1996; Herskin, 1999). In larvae, acclimation to respirometer has not been commonly tested and it is not clear if it really occurs. Larvae of F. paulensis exhibited decrease in VO2 with time after being placed in chamber. Higher VO2 registered in ‘nonacclimated larvae’ may also be attributed to the calorigenic effect of food (SDA) (Du Preez et al., 1992; Rosas et al., 1996) since the difference between initial and stabilized VO2 of F. paulensis was equivalent to SDA values reported for Metapenaeus ensis larvae (Chu and Ovsianico-Koulikowsky, 1994). The time for the stabilization of VO2 also coincided with previous observations of empty guts in larvae after 2 h absence of food. Oxygen levels were maintained higher than 80% saturation since lower concentrations may affect metabolic rates in larval crustaceans (Dawirs, 1983). At the proposed stocking density, nauplius and protozoea reached 80% saturation in a shorter time compared to mysis and the first postlarva (see Section 3). Though these stages were stocked at lower biomass-volume ratios (12.1–22.8 mg mly1 N III-PZIII vs. 26.7–30.6 mg mly1in M IPL I), elevated metabolic rates of early larva produced a faster decrease in oxygen concentration. The decline in metabolic rates throughout the
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larval development has been reported for crustaceans (Anger and Jacobi, 1985; Chu and Ovsianico-Koulikowsky, 1994; Lemos and Phan, 2001a) as well as for other invertebrate species (Jaeckle and Manahan, 1989; Hoegh-Guldberg and Manahan, 1995). It can be mainly attributed to: (1) the decrease in surface: volume ratio that limits supply or removal processes, (e.g. gas exchange, digestive ability) (Childress and Somero, 1990); (2) the ontogenetic shift in life style, as in penaeid shrimp, from the typical planktonic (nauplius and protozoea), passing through a transitional phase (mysis and early postlarva) until the full adoption of benthic existence (Iwai, 1978). These bioenergetic shifts are common through the development of invertebrate larvae and must be considered for the quantification of metabolic rates. The present method produced reasonable values of oxygen consumption and ammonia-N excretion in shrimp and mussel early stages, and can be further applied to the development of other larval invertebrates, especially those including planktonic forms. The accuracy of values obtained as denoted by the coefficient of variation can be considered comparable to other studies of metabolic rates in larval invertebrates (Quetin and Ross, 1989; Hoegh-Guldberg and Emlet, 1997; Anger et al., 1998; Richmond and Woodin, 1999). Individual oxygen consumption rates (VO2) of larval F. paulensis were close to findings on Metapenaeus ensis at comparable temperature and salinity conditions (Chu and Ovsianico-Koulikowsky, 1994). Values of F. paulensis were similar to M. ensis in embryo and PZ I (0.01 and 0.1 mg O2 indy1 hy1, respectively), and belonged to the same order of magnitude in nauplius, M III and postlarva (0.02, 0.38 and 0.65 mg O2 indy1 hy1, respectively). If oxygen consumption rate is standardized on a Q10s 3.8 (Sprung, 1984), present results of P. perna VO2 (0.85–3.02 mg O2 indy1 hy1) stay in the range reported for larval Mytilus edulis, as 0.76– 5.76 mg O2 indy1 hy1 (Sprung, 1984) and 0.50– 1.25 mg O2 indy1 hy1 (Wang and Widdows, 1991). Individual ammonia-N excretion of larval shrimp also exhibited comparable results with embryo, nauplius, PZ III and M III of M. ensis with 0.001, 0.003, 0.012 and 0.025 mg N-NH3 indy1 hy1, respectively, (Chu and Ovsianico-Koulikowsky, 1994). The present protocol combines methodological simplicity with the precision of Winkler’s titration in determining oxygen content, considered more
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reliable than polarographic oxygen sensors that may underestimate larval respiration (Shilling et al., 1996; Marsh and Manahan, 1999). Since the amount of energy expended in metabolism as measured by oxygen consumption is a major parameter in the energy budget of larval invertebrates (Mootz and Epifanio, 1974; Hoegh-Guldberg and Emlet, 1997; Lemos and Phan, 2001b), a correct quantification of metabolic rates contributes to determine the actual amount of energy to succeed throughout early ontogenetic development. References Anger, K., Jacobi, C.C., 1985. Respiration and growth of Hyas araneus L. larvae (Decapoda: Majidae) from hatching to metamorphosis. J. Exp. Mar. Biol. Ecol. 88, 257–270. Anger, K., Spivak, E., Luppi, T., 1998. Effects of reduced salinities on development and bioenergetics of early larval shore crab, Carcinus maenas. J. Exp. Mar. Biol. Ecol. 220, 287–304. Bhatnagar, K.M., Crisp, D.J., 1965. The salinity tolerance of nauplius larvae of cirripedes. J. Anim. Ecol. 34, 418–419. Buttle, L.G., Uglow, R.F., Cowx, I.G., 1996. The effect of emersion and handling on the nitrogen excretion rates of Clarias gariepinus. J. Fish Biol. 49, 693–701. Childress, J.J., Somero, G.N., 1990. Metabolic scaling: a new perspective based on scaling of glycolytic enzyme activities. Amer. Zool. 30, 161–173. Chu, K.H., Ovsianico-Koulikowsky, N.N., 1994. Ontogenetic changes in metabolic activity and biochemical composition in the shrimp, Metapenaeus ensis. J. Exp. Mar. Biol. Ecol. 183, 11–26. Dall, W., Hill, B.J., Rothlisberg, P.C., Staples, D.J., 1990. The biology of Penaeidae. Adv. Mar. Biol. 27, 1–489. 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. 69, 105–128. Du Preez, H., Chen, H.-Y., Hsieh, C.-S., 1992. Apparent specific dynamic action of food in the grass shrimp, Penaeus monodon Fabricius. Comp. Biochem. Physiol. A 103, 173–178. Fox, H.M., Wingfield, C.A., 1938. A portable apparatus for the determination of oxygen dissolved in small volume of water. J. Exp. Biol. 15, 437–445. Gnaiger, E., 1983. Calculation on energetic and biochemical equivalents of respiratory oxygen consumption. In: Gnaiger, E., Forstner, H. (Eds.), Polarographic Oxygen Sensors. Springer, Berlin, pp. 337–345. Grasshoff, K., 1976. Methods of Seawater Analysis. Verlag Chemie, Weinhein. Herskin, J., 1999. Effects of social and visual contact on the oxygen consumption of juvenile sea bass measured by computerized intermittent respirometry. J. Fish Biol. 55, 1075–1085. Hoegh-Guldberg, O., Emlet, R.B., 1997. Energy use during the development of a lecithotrophic and a planktotrophic echinoid. Biol. Bull. 192, 27–40.
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Hoegh-Guldberg, O., Manahan, D.T., 1995. Coulometric measurement of oxygen consumption during development of marine invertebrate embryos and larvae. J. Exp. Biol. 198, 19–30. ´ Iwai M., 1978. Desenvolvimento larval e pos-larval de Penaeus ´ (Melicertus) paulensis Perez-Farfante 1967 (Crustacea, ˜ ˆ Decapoda) e o ciclo de vida dos camaroes do genero ˜ centro-sul do Brasil. Ph.D. Thesis, UniPenaeus da regiao ˜ Paulo, IBUSP, Sao ˜ Paulo, Brazil, 138p. versity of Sao Jaeckle, W.B., Manahan, D.T., 1989. Growth and energy imbalance during development of the lecithotrophic molluscan larva Haliotis rufescens. Biol. Bull. 177, 237–246. Jones, D.R., Randall, D.J., 1978. The respiratory and circulatory system during exercise. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, Vol. 7. Academic Press, New York, pp. 425–501. Koroleff, F., 1983. Determination of ammonia. In: Grasshoff, K., Ehrhardt, M., Kremling, K. (Eds.), Methods of Seawater Analysis. 2nd ed. Verlag Chemie, Weinheim, pp. 150–157. Lemos, D., Phan, V.N., 2001a. Ontogenetic variation in metabolic rates, biochemical composition and energy content during the early life stages of Farfantepenaeus paulensis (Crustacea: Decapoda: Penaeidae). Mar. Biol. 138, 985–997. Lemos, D., Phan, V.N., 2001b. Energy partitioning into growth, respiration, excretion and exuvia during larval development of the shrimp Farfantepenaeus paulensis. Aquaculture 199, 133–145. Marsh, A.G., Manahan, D.T., 1999. A method for accurate measurements of the respiration rates of marine invertebrate embryos and larvae. Mar. Ecol. Prog. Ser. 184, 1–10. Mayzaud, P., Conover, R.J., 1988. O:N atomic ratio as a tool to describe zooplankton metabolism. Mar. Ecol. Prog. Ser. 45, 289–302. Mootz, C.A., Epifanio, C.E., 1974. An energy budget for Menippe mercenaria larvae fed Artemia nauplii. Biol. Bull. 146, 44–55. Primavera, J.H., Posadas, R.A., 1981. Studies on the egg quality of Penaeus monodon Fabricius, based on morphology and hatching rates. Aquaculture 22, 269–277. Quetin, L.B., Ross, R.M., 1989. Effects of oxygen, temperature and age on the metabolic rate of the embryos and early
larval stages of the Antarctic krill Euphausia superba Dana. J. Exp. Mar. Biol. Ecol. 125, 43–62. Richmond, C.E., Woodin, S.A., 1999. Effect of salinity reduction on oxygen consumption by larval estuarine invertebrates. Mar. Biol. 134, 259–267. ´ y desarollo Rojas, A.V., Martinez, R., 1967. Reproduccion ´ comestible de Venezuela larval experimental del mejillon Perna perna (Linnaeus, 1758). Bol. Inst. Oceanog. Univ. Oriente 6, 266–285. Rosas, C., Sanchez, A., Diaz, E., Soto, L.A., Gaxiola, G., Brito, R., 1996. Effect of dietary protein level on apparent heat increment and post-prandial nitrogen excretion of Penaeus setiferus, P. schmitti, P. duorarum and P. notialis postlarvae. J. World Aquacult. Soc. 27 (1), 92–102. Scholander, P.F., Claff, C.L., Sveinsson, S.L., Scholander, S.I., 1952. Respiratory studies of single cells. III Oxygen consumption during cell division. Biol. Bull. 102, 185–199. Shilling, F.M., Hoegh-Guldberg, O., Manahan, D.T., 1996. Sources of energy for increased metabolic demand during metamorphosis of the abalone Haliotus rufescens (Mollusca). Biol. Bull. 191, 402–412. Sprung, M., 1984. Physiological energetics of mussel larvae (Mytilus edulis). III. Respiration. Mar. Ecol. Prog. Ser. 18, 171–178. Vinatea, L., Olivera, A., Andreatta, E., Beltrame, E., Petersen, ´ comercial de larvas de R., Derner, R., 1993. Produccion Penaeus paulensis y Penaeus schmitti en el sur del Brasil. ´ In: ABCC, (Ed.), Anais do Quarto Simposio Brasileiro ˜ MCR Aquicultura. Joao ˜ Pessoa, sobre Cultivo de Camarao, Brazil, pp. 399–414. Wang, W.X., Widdows, J., 1991. Physiological responses of mussel larvae Mytilus edulis to environmental hypoxia and anoxia. Mar. Ecol. Prog. Ser. 70, 223–236. Wyban, J., Cheng, S.L., Sweeney, J., Richards Jr, W.K., 1987. Observation on development of a maturation system for Penaeus vannamei. J. World Aquacult. Soc. 18, 198–200. Zar, J.H., 1984. Biostatistical Analysis. 2nd ed. Prentice Hall, Englewood Cliffs, New Jersey. Zeuthen, E., 1943. A cartesian diver micro respirometer with a gas volume of 0.1 ml. CRT Lab. Carlsberg. Ser. Chim. 24, 479–518.
Simultaneous measurements of oxygen consumption and ammonia-N excretion in embryos and larvae of marine invertebrates D. Lemosa,*, R.L.V. Jorgeb, V.N. Phana a
b
´ ˜ Paulo, C.P. P.O. Box 66149, Sao ˜ Paulo, SP 05389-900, Brazil Instituto Oceanografico, University of Sao ˜ Carlos, University of Sao ˜ Paulo, C.P. P.O. Box 292, Sao ˜ Carlos, SP 13560-970, Brazil Escola de Engenharia de Sao Received 18 November 2002; received in revised form 2 June 2003; accepted 2 June 2003
Abstract The quantification of oxygen consumption and ammonia-N excretion rates is essential in determining energy requirements for development of larval invertebrates. In larval energetics, there is a need for accurate and uncomplicated techniques to quantify metabolic rates. A method for simultaneous measurements of oxygen and ammonia-N concentrations is presented. It employs sealed respirometric chambers (ca. 30 ml) in which embryos and larvae are incubated. Analysis is carried out in end-point samples by Winkler’s titration and indophenol-blue for oxygen and ammonia-N, respectively. Water is sampled into volume-calibrated glass syringes and oxygen consumption and ammoniaN excretion rates were determined by the difference between experimental and control (no animals) units. The method was successfully used to measure metabolic rates in embryo and larval stages of the shrimp Farfantepenaeus paulensis and in veliger of the mussel Perna perna. The accuracy denoted by the coefficient of variation is comparable to previous results on larval metabolic rates. A biomass: volume (mg mly1 ) is proposed to extend its application to further species of marine invertebrates. The method is simple to operate, involves non-expensive material and is portable enough for field work. A substantial number of replicates can be analyzed at the same time and O:N ratio, an indicator of the catabolized substrate, can be calculated. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Embryo; Excretion; F. paulensis; Larvae; Mussel; Oxygen-consumption; P. perna; Shrimp
1. Introduction Energy requirements for embryonic and larval development of marine invertebrates can be determined based on correct measurements of oxygen consumption and ammonia-N excretion rates. In hatcheries, early ontogenetic stages are often maintained in high densities and data on oxygen demand and ammonia-N release rates are necessary for a correct dimensioning of structures and routine of cultivation. Several methods have been devel*Corresponding author. Fax: q55-11-3091-6607. E-mail address: [email protected] (D. Lemos).
oped for the quantification of oxygen consumption in embryos and larvae of invertebrates such as micro-Cartesian divers (Zeuthen, 1943; Scholander et al., 1952), Winkler’s titration (Bhatnagar and Crisp, 1965) and polarographic oxygen sensors (Gnaiger, 1983; Hoegh-Guldberg and Emlet, 1997) adapted to micro-respiration chambers. Some of them are rather complicated and difficult to handle in field conditions, being not routinely used (Marsh and Manahan, 1999). Few methods have been described for simultaneous determination of oxygen consumption and ammonia-N that enables the calculation of O:N, an indicator of the catab-
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1095-6433(03)00163-6
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olized substrate (Mayzaud and Conover, 1988). The accuracy of the technique is possibly the most important aspect to consider. For instance, continuous measurements of oxygen consumption by polarographic oxygen sensors may underestimate respiration rates compared to Winkler’s titration (Shilling et al., 1996; Marsh and Manahan, 1999). The development of alternative and accurate techniques for determination of larval metabolic rates is thus highly desirable. A method for determination of oxygen consumption and ammonia-N excretion rates in embryos and larval invertebrates is here described. Evaluation of oxygen and ammonia-N were carried out in sealed respirometers through analysis of end-point samples. The technique combines the use of simple equipment with the precision of Winkler’s and indophenol-blue (Koroleff, 1983) procedures for determination of oxygen and ammonia, respectively. It also allows a substantial number of replicates at the same time. The detailed protocol is for first time introduced and efficacy is demonstrated by data on metabolic rates of larval shrimp F. paulensis (Lemos and Phan, 2001a) and mussel P. perna. 2. Material and methods 2.1. Experimental animals Adult shrimps F. paulensis were obtained from the commercial fishery at Santa Catarina State coast, Brazil (278189 Lat S, 488239 Long W). After acclimation to culture conditions, females (33.9"4.7 g wet mass) were induced to sexual maturity by unilateral eyestalk ablation (Wyban et al., 1987), and individually isolated in 500-l fiberglass tanks. Spawning occurred at night, and viable eggs (ca. 200 000 per female) were stocked in cylindrical-conical tanks, hatching 12 to 14 h later at 26"1 8C. Larval development of F. paulensis presents six naupliar (N I to VI), three protozoeal (PZ I to III), and three mysid (M I to III) stages (Iwai, 1978). At nauplius V-VI, larvae were moved to 50 000 l tanks, and reared according to commercial practices at 26"1 8C and 34"1‰ S (Vinatea et al., 1993). Larvae were initially stocked at 100 ind ly1 with strong aeration and a 40–60% water renewal daily. Exogenous food was supplied from the stage PZ I and was made of the diatom Chaetoceros calcitrans (80 000 cells mly1) and artificial plankton (Nippai Shrimp Feed Inc.,
Japan; 0.03 mg larvay1 dayy1, 30 mm particlesize). From PZ III, freshly hatched nauplii of Artemia sp. (5 ind larvay1 dayy1) were added to the diet. The Artemia sp. ration was set to 15 nauplii larvay1 dayy1 between M I and M III. After metamorphosis, postlarvae (PL) were fed microalgae with increasing amounts of artificial plankton and brine shrimp nauplii. Metabolic rates of larval shrimp were determined at 34"1‰ and 26"0.5 8C. Samples of pooled individuals at same developmental stage were selected from a tank containing nine synchronized spawns. A homogeneous sample was characterized when )80% of individuals belonged to the same stage, the remaining individuals differing by only a single stage. Embryos were sampled at the ‘early nauplius’ embryonic stage, 10 to 12 h after spawning (Primavera and Posadas, 1981). Oxygen consumption and ammonia-N excretion were then determined in embryo, N III, PZ I and in each subsequent larval stage until metamorphosis. The first postlarval instar was verified after the molt of metamorphosis, sorted by the number of rostral teeth combined to 6th segment setae (Iwai, 1978). Individuals from different stages were chosen near to the intermolt period that comprises 7–30% of the total molt cycle since the last ecdysis (Dall et al., 1990). ˜ Sebastiao ˜ Mussels P. perna were caught at Sao Island rocky shores (238549 Lat S, 458279 Long W). Spawning was obtained after keeping separated males and females exposed to air at 17.0"2.0 8C for 15 h followed by immersion in UV sterilized seawater at 28.5"2.5 8C and 34"1‰. Sperm and oocytes were separately washed through a sequence of 100 to 22 mm mesh size nets in order to clear from debris. Oocytes were fertilized by the addition of fresh sperm followed by gently shaking and incubation at 24"0.5 8C with smooth aeration. After 27 h, unfed veliger individuals (82.05"6.67 mm) were separated through 100 to 50 mm nets and counted in Sedgewick–Rafter chambers under optical microscope. Experiments were carried out in 34"1‰ and 24"0.5 8C. Larval stages were identified according to Rojas and Martinez (1967). Replicates of pooled shrimp or mussel larvae were rinsed with distilled water, dried with filter paper and separated to mass determination. Shrimp samples were dried for 48 h at 70 8C, and weighed on a Cahn C-31 microbalance to the nearest 0.1
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required for animals to acclimate in the chamber was previously established by measures of oxygen consumption of shrimp protozoea since the beginning of the confinement. After the acclimation period, the net-cover was removed and chambers were carefully closed as the excess water escaped through the cover orifice, expelling the remaining air in between water and cover surface. The plastic tablet was laid over the external excess water covering the orifice and generating a tension to avoid water contact with air. Five to ten replicate experimental chambers were accompanied by 5 controls (no animals) in order to detect any possible oxygen variation due to biological or chemical demand. Units were then placed in a temperature controlled water bath during the incubation. Initial oxygen concentration was near to saturation at that temperature and salinity conditions (Grasshoff, 1976). The time of incubation varied among developmental stages and oxygen levels were not allowed to reach less than 80% saturation (Dawirs, 1983). Fig. 1. Respirometric chamber used in measurements of oxygen consumption ammonia-N excretion of embryos and larvae of marine invertebrates.
mg. After 48 h at 80 8C, mussel larvae were weighed in a Mettler Toledo balance (precision 0.1 mg). 2.2. Respirometric chambers Individuals were placed in cylindrical plastic chambers of ca. 30 ml with a plastic cover that provide hermetical sealing (Fig. 1). An orifice of 1.5 mm placed in the center of the cover allowed the elimination of air bubbles from inside the chamber prior to closing. A plastic tablet covered the orifice isolating inside water by holding a tension between the tablet and the water in the orifice. In our experiments, 35 mm translucent photographic film containers were used showing inert, adequate for handling, ease to obtain and maintain constant volume. Chambers were individually identified and exact volume was determined gravimetrically. A known number of individuals were put into the chambers and filled with clear water to the top of the container. Respirometric units (identified containers) were covered by an adapted plastic cover with a net of adequate mesh size to avoid animal escape, and submitted to flow through clear seawater for acclimation. The time
2.3. Water sampling and analysis Water was sampled by using 10 ml glasssyringes (Becton and Dickinson) equipped with plastic nozzles. Samples contained 8 ml of water in the barrel plus the volume in the dead space in the nozzle. The volume of the dead space in the nozzle was chemically determined in each syringe by filling it with 0.025 N potassium iodate following the addition of 1% (wyv) potassium iodide and the titration against 0.01 N sodium thiosulfate (Fox and Wingfield, 1938). Volume calibrated syringes were used in sampling for oxygen determination. A plastic cannula attached to the tip of the nozzle was introduced into the respirometric chamber by the cover orifice. Two milliliters were drawn up into the barrel and washed out to eliminate bubbles from inside barrel and nozzle. Samples were taken carefully until the mark of 8 ml. Reagents for oxygen determination by the Winkler method wmanganic (0.4 ml) and alkaline (0.8 ml) solutions, phosphoric acid (0.8 ml)x were drawn up directly into the syringes up to the mark of 10 ml. The plunger was locked after covering the nozzle with a plastic nozzle-shaped jacket. Samples were titrated against 0.01 N sodium thiosulfate by using a portable micrometer burette (Gilson Instruments) to the nearest 0.001 ml. The oxygen concentration in samples was calculated
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Table 1 Salt effect factor (SF) to correct ammonia-N values in seawater (after Koroleff, 1983) Salinity (‰) 0 SF
5
10
15
20
25
30
in oxygen or ammonia-N contents. Oxygen consumption and ammonia-N excretion rates were determined by the difference between experimental and control units, and were expressed as individual (mg indy1 hy1) and dry mass-specific (mg gy1 DW hy1) rates. Atomic O:N ratios could be calculated by dividing values of oxygen consumed by the nitrogen excreted in each developmental stage (Mayzaud and Conover, 1988).
35
1.00 1.03 1.06 1.09 1.14 1.18 1.22 1.25
as: wO2x (mg ly1)s8000=N=HyV or wO2x (ml ly1)s5600=N=HyV
2.4. Statistical analysis Differences among means were detected by one way ANOVA followed by Tukey’s multicomparison test. When data did not follow normal distribution, differences were verified by the non-parametric Kruskal–Wallis analysis followed by mean comparison of Nemenyi. Results were considered significant at P-0.05 (Zar, 1984).
where Nsvolume of sodium thiosulfate expended; Hsnormality of sodium thiosulfate; Vssample volume (barrelqnozzle). The solution of sodium thiosulfate used in titration was standardized at regular intervals to determine its exact normality. After sampling for oxygen content, 10 ml were taken from respirometric chambers by glass syringes and put in amber flasks for determination of ammonia-N content. Washing samples (2 ml) were drawn up as for oxygen determination to eliminate bubbles from inside barrel. Solutions of phenoldisodium nitroprusside (R1) and trisodium citratesodium hypochlorite (R2) were added for the formation of indophenol blue (Koroleff, 1983). Reagents were standardized with solution of ammonia chloride dissolved in ammonia-free purified water (Milli Q娃 –Millipore). When analyzing seawater, dissolved salts reduce the indophenol blue produced so that values were corrected by the salt effect factor (Table 1). The eventual presence of organisms drawn up through water samples did not result in difference
3. Results The number of shrimp larvae incubated in respirometric chambers varied according to individual dry mass (Table 2). Five-hundred shrimp embryos were necessary to produce changes in oxygen and ammonia-N concentration while the same was reached by 8 to 130 individuals in the following larval stages. Metabolic rate of nauplius III (N III) with reduced mass compared to embryo was detected in groups of 130 individuals. In the following stages, the number of individuals pooled decreased with the increment in dry mass. Incubation time for each stage varied between 90 (protozoea I and II) and 165 min (N III) and was not related to the number of individuals in chamber. For mussel, a higher number of veliger (4500
Table 2 Age, dry mass (DW), number of individuals, incubation time and biomass: volume ratio in closed respirometers for determination of oxygen consumption and ammonia-N excretion during the early life stages of F. paulensis at 26"0.5 8C. Nsnauplius, PZsprotozoea, Msmysis and PLspostlarva. Roman numerals denote the number of molts in stage Stage
Age (days after spawning)
DW (mg)
Individuals in chamber
Incubation time (min)
Biomass: volume (mg mly1)
Embryo N III PZ I PZ II PZ III MI M II M III PL I
0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5
4.1 2.8 6.6 13.7 24.0 53.5 81.3 91.7 108.7
500 130 80 50 20 15 10 10 8
120 165 90 90 120 150 130 140 120
68.3 12.1 17.6 22.8 16.0 26.7 27.1 30.6 29.0
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Table 3 Age, dry weight (DW), number of individuals, incubation time and biomass: volume ration in closed respirometers for determination of oxygen consumption and ammonia-N excretion in veliger of P. perna at 24"0.5 8C Stage
Age (days after spawning)
DW (mg)
Individuals in chamber
Incubation time (min)
Biomass: volume (mg mly1)
Veliger
1.12
0.0984
4500
300
14.8
further determinations of metabolic rates in other larval species. Acclimation to respirometers was determined by oxygen consumption rates since the moment shrimp larva was placed in the chamber (Fig. 2). Individual oxygen consumption (VO2) of PZ III decreased significantly after 20 min incubation (P-0.05) and stabilized after 100 min in PZ III. In the following experiments, animals were kept for a minimum of 100 min in flow-through seawater prior to close respirometers. The time of incubation required to 80% oxygen saturation varied among shrimp larval stages, extending from ca. 190 min in embryo, N III and PZ II (P)0.05) to 215 min in M I and 310 min in PL I. Individual rates of oxygen consumption (VO2) increased significantly throughout shrimp development (P-0.05) (Table 4). In contrast, massspecific rates (QO2) varied significantly throughout ontogenetic stages. Higher rates were observed in stages N III and PZ I, 30.0 and 24.0 mg O2 gy1 DW hy1, respectively. Mussel veliger presented reduced VO2 and individual ammonia-N excretion compared to shrimp stages (Table 5). However, in mass-specific basis, veliger showed QO2 as high as shrimp protozoeal stages. Individual ammonia-N excretion increased through the majority of shrimp developmental stages, excepting the intervals M I-M II and M III-PL I that presented reduced rates (Table 4).
Fig. 2. Individual oxygen consumption related to time of acclimation to sealed repirometers in protozoea III (PZ III) of F. paulensis at 26"0.5 8C. Results expressed as means, error barssS.D. Different letters denote significant differences (P) 0.05). (Data from Lemos and Phan, 2001a).
ind) was necessary for the determination of metabolic rate as a longer incubation time (Table 3). Though experiments were carried in different temperatures, the biomass-volume ratio (by dividing the total dry mass of individuals by the chamber volume—mg mly1) was 68.3 for benthic embryo and ranged from 12.1 to 30.6 for planktonic larval shrimp and mussel (Tables 2 and 3). At the same temperature, early larval stages required lower biomass-ratios. The biomass–volume ratio can contribute to establish the stocking density in
Table 4 Individual and mass-specific rates of oxygen consumption and ammonia-N excretion, and O:N ratios during the early life stages of the shrimp F. paulensis at 26"0.5 8C. Results expressed as means (S.D. in parentheses). (Data from Lemos and Phan, 2001a) Stage
mg O2 indy1 hy1
Embryo N III PZ I PZ II PZ III MI M II M III PL I
0.012 0.083 0.158 0.212 0.362 0.667 0.859 0.986 1.209
(0.002) (0.015) (0.007) (0.029) (0.108) (0.170) (0.089) (0.098) (0.322)
mg N-NH3 indy1 hy1 0.0012 0.0019 0.0048 0.0103 0.0126 0.0182 0.0112 0.0193 0.0096
(0.00041) (0.00029) (0.00031) (0.0047) (0.0029) (0.0026) (0.0037) (0.0051) (0.0013)
mg O2 g DWy1 hy1 7.6 30.0 24.0 15.5 15.1 12.5 10.6 10.7 11.1
(1.28) (5.40) (1.10) (2.10) (4.49) (3.18) (1.10) (1.07) (2.97)
mg N-NH3 g DWy1 hy1 0.30 0.68 0.73 1.02 0.52 0.34 0.14 0.21 0.09
(0.10) (0.11) (0.05) (0.08) (0.12) (0.05) (0.05) (0.05) (0.01)
O:N 6.5 27.5 19.4 9.0 18.7 17.9 57.4 66.6 82.4
(2.9) (7.3) (1.7) (1.1) (3.1) (2.6) (6.5) (10.5) (8.4)
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Table 5 Individual and mass-specific rates of oxygen consumption and ammonia-N excretion, and O:N ratio of larval mussel P. perna at 24"0.5 8C. Results expressed as means (S.D. in parentheses) Stage
ng O2 indy1 hy1
ng N-NH3 indy1 hy1
mg O2 g DWy1 hy1
mg N-NH3 g DWy1 hy1
O:N
Veliger
1.96 (0.62)
0.443 (0.081)
19.87 (6.22)
4.50 (0.83)
3.7 (1.2)
Mass-specific rates of ammonia-N excretion also varied significantly during development (P0.05). Individual excretion rates were reduced in larval mussel compared to shrimp while massspecific rates were much higher (Table 5). As a general trend, shrimp development was characterized by low O:N values between PZ I and M I, and high values between M II and PL I. Mussel veliger showed decreased O:N value compared to shrimp larvae (Table 5). Data on shrimp oxygen consumption, ammonia-N excretion and O:N performed a mean coefficient of variation (CV) of 17, 23 and 17%, respectively. Variation on rates of larval mussel showed a CV of 31, 18 and 32% for oxygen consumption, ammonia-N excretion and O:N, respectively. 4. Discussion Oxygen consumption is a major energy expense throughout larval development of marine invertebrates and requires precise measurement. However, due to the reduced size of individuals and the small amount of oxygen consumed, sensitive and accurate techniques are necessary. The present method enables simultaneous measurements of oxygen consumption and ammonia-N excretion of invertebrate embryos and larvae by using simple and inexpensive material, with reduced dimensions that can be easily transported. The experimental routine is rather uncomplicated and permits application after a few hours training. It also allows a substantial number of replicates at the same time and slight variations in oxygen and ammonia-N concentrations can be detected. Incubation period mostly depends on metabolic rates and stocking density of individuals. Planktonic larvae must be arranged as to enable freeswimming in the chamber and densities can be calculated from biomass-volume ratios (Tables 2 and 3). At the same temperature, early stages require lower biomass-ratios compared to advanced larval stages. The number of larval shrimp or mussel stocked may eventually be altered from that presented depending on experi-
mental purpose and culture conditions, (e.g. temperature gradient, exposure to pollutants). Experiments with benthic embryos that remain deposited over the bottom during incubation may produce vertical stratification of oxygen and ammonia-N concentration. This concern can be minimized through the collection of a large sample volume as it has been taken for oxygen (ca. 8 ml) and ammonia-N (10 ml) regarding total chamber capacity. Acclimation to respirometric chambers has been reported for juvenile and adult marine animals by decrease in activity and oxygen consumption (Jones and Randall, 1978). Accordingly, animals previously subjected to stresses of handling and space limitation adapt to experimental chambers after being submitted to certain conditions as clear and oxygenated water, absence of external movement and visual disturbance (Buttle et al., 1996; Herskin, 1999). In larvae, acclimation to respirometer has not been commonly tested and it is not clear if it really occurs. Larvae of F. paulensis exhibited decrease in VO2 with time after being placed in chamber. Higher VO2 registered in ‘nonacclimated larvae’ may also be attributed to the calorigenic effect of food (SDA) (Du Preez et al., 1992; Rosas et al., 1996) since the difference between initial and stabilized VO2 of F. paulensis was equivalent to SDA values reported for Metapenaeus ensis larvae (Chu and Ovsianico-Koulikowsky, 1994). The time for the stabilization of VO2 also coincided with previous observations of empty guts in larvae after 2 h absence of food. Oxygen levels were maintained higher than 80% saturation since lower concentrations may affect metabolic rates in larval crustaceans (Dawirs, 1983). At the proposed stocking density, nauplius and protozoea reached 80% saturation in a shorter time compared to mysis and the first postlarva (see Section 3). Though these stages were stocked at lower biomass-volume ratios (12.1–22.8 mg mly1 N III-PZIII vs. 26.7–30.6 mg mly1in M IPL I), elevated metabolic rates of early larva produced a faster decrease in oxygen concentration. The decline in metabolic rates throughout the
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larval development has been reported for crustaceans (Anger and Jacobi, 1985; Chu and Ovsianico-Koulikowsky, 1994; Lemos and Phan, 2001a) as well as for other invertebrate species (Jaeckle and Manahan, 1989; Hoegh-Guldberg and Manahan, 1995). It can be mainly attributed to: (1) the decrease in surface: volume ratio that limits supply or removal processes, (e.g. gas exchange, digestive ability) (Childress and Somero, 1990); (2) the ontogenetic shift in life style, as in penaeid shrimp, from the typical planktonic (nauplius and protozoea), passing through a transitional phase (mysis and early postlarva) until the full adoption of benthic existence (Iwai, 1978). These bioenergetic shifts are common through the development of invertebrate larvae and must be considered for the quantification of metabolic rates. The present method produced reasonable values of oxygen consumption and ammonia-N excretion in shrimp and mussel early stages, and can be further applied to the development of other larval invertebrates, especially those including planktonic forms. The accuracy of values obtained as denoted by the coefficient of variation can be considered comparable to other studies of metabolic rates in larval invertebrates (Quetin and Ross, 1989; Hoegh-Guldberg and Emlet, 1997; Anger et al., 1998; Richmond and Woodin, 1999). Individual oxygen consumption rates (VO2) of larval F. paulensis were close to findings on Metapenaeus ensis at comparable temperature and salinity conditions (Chu and Ovsianico-Koulikowsky, 1994). Values of F. paulensis were similar to M. ensis in embryo and PZ I (0.01 and 0.1 mg O2 indy1 hy1, respectively), and belonged to the same order of magnitude in nauplius, M III and postlarva (0.02, 0.38 and 0.65 mg O2 indy1 hy1, respectively). If oxygen consumption rate is standardized on a Q10s 3.8 (Sprung, 1984), present results of P. perna VO2 (0.85–3.02 mg O2 indy1 hy1) stay in the range reported for larval Mytilus edulis, as 0.76– 5.76 mg O2 indy1 hy1 (Sprung, 1984) and 0.50– 1.25 mg O2 indy1 hy1 (Wang and Widdows, 1991). Individual ammonia-N excretion of larval shrimp also exhibited comparable results with embryo, nauplius, PZ III and M III of M. ensis with 0.001, 0.003, 0.012 and 0.025 mg N-NH3 indy1 hy1, respectively, (Chu and Ovsianico-Koulikowsky, 1994). The present protocol combines methodological simplicity with the precision of Winkler’s titration in determining oxygen content, considered more
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reliable than polarographic oxygen sensors that may underestimate larval respiration (Shilling et al., 1996; Marsh and Manahan, 1999). Since the amount of energy expended in metabolism as measured by oxygen consumption is a major parameter in the energy budget of larval invertebrates (Mootz and Epifanio, 1974; Hoegh-Guldberg and Emlet, 1997; Lemos and Phan, 2001b), a correct quantification of metabolic rates contributes to determine the actual amount of energy to succeed throughout early ontogenetic development. References Anger, K., Jacobi, C.C., 1985. Respiration and growth of Hyas araneus L. larvae (Decapoda: Majidae) from hatching to metamorphosis. J. Exp. Mar. Biol. Ecol. 88, 257–270. Anger, K., Spivak, E., Luppi, T., 1998. Effects of reduced salinities on development and bioenergetics of early larval shore crab, Carcinus maenas. J. Exp. Mar. Biol. Ecol. 220, 287–304. Bhatnagar, K.M., Crisp, D.J., 1965. The salinity tolerance of nauplius larvae of cirripedes. J. Anim. Ecol. 34, 418–419. Buttle, L.G., Uglow, R.F., Cowx, I.G., 1996. The effect of emersion and handling on the nitrogen excretion rates of Clarias gariepinus. J. Fish Biol. 49, 693–701. Childress, J.J., Somero, G.N., 1990. Metabolic scaling: a new perspective based on scaling of glycolytic enzyme activities. Amer. Zool. 30, 161–173. Chu, K.H., Ovsianico-Koulikowsky, N.N., 1994. Ontogenetic changes in metabolic activity and biochemical composition in the shrimp, Metapenaeus ensis. J. Exp. Mar. Biol. Ecol. 183, 11–26. Dall, W., Hill, B.J., Rothlisberg, P.C., Staples, D.J., 1990. The biology of Penaeidae. Adv. Mar. Biol. 27, 1–489. 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. 69, 105–128. Du Preez, H., Chen, H.-Y., Hsieh, C.-S., 1992. Apparent specific dynamic action of food in the grass shrimp, Penaeus monodon Fabricius. Comp. Biochem. Physiol. A 103, 173–178. Fox, H.M., Wingfield, C.A., 1938. A portable apparatus for the determination of oxygen dissolved in small volume of water. J. Exp. Biol. 15, 437–445. Gnaiger, E., 1983. Calculation on energetic and biochemical equivalents of respiratory oxygen consumption. In: Gnaiger, E., Forstner, H. (Eds.), Polarographic Oxygen Sensors. Springer, Berlin, pp. 337–345. Grasshoff, K., 1976. Methods of Seawater Analysis. Verlag Chemie, Weinhein. Herskin, J., 1999. Effects of social and visual contact on the oxygen consumption of juvenile sea bass measured by computerized intermittent respirometry. J. Fish Biol. 55, 1075–1085. Hoegh-Guldberg, O., Emlet, R.B., 1997. Energy use during the development of a lecithotrophic and a planktotrophic echinoid. Biol. Bull. 192, 27–40.
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