Ontogenetic development of digestive enzyme activities in larval walleye pollock, Theragra chalcogramma. Received: 7 October 1994 / 5 December 1994.
Marine Biology(1995) 122:177-186
9 Springer-Verlag 1995
Y. Oozeki. K. M. Bailey
Ontogenetic development of digestive enzyme activities in larval walleye pollock, Theragrachalcogramma
Received: 7 October 1994 / 5 December 1994
Activities of digestive enzymes trypsin, amy- val growth rate results in a shorter duration of the larval lase and lipase in laboratory-reared walleye pollock, The- period and consequently, higher survival (Miller et al. ragra chalcogramma, were measured from hatching to Day 1988). 39 (just before notochord flexion) in 1993. All measureWalleye pollock, Theragra chalcogramma (Pallas), is ments were conducted individually or semi-individually widely distributed and is the most abundant commercial (groups of two larvae of the same standard length). Close species in the northeastern Pacific Ocean (Bakkala et al. relationships between digestive enzyme activities and 1986). Significant differences in growth rates have been morphological development of digestive organs were ob- observed between laboratory-reared (Bailey and Stehr served. Activities of trypsin and lipase were low during the 1986, 1988) and wild-caught walleye pollock larvae (Waltransition period from endogenous to exogenous energy. line 1985; Kendall et al. 1987; Yoklavich and Bailey 1988, Amylase activity was constant with large variance during 1990). Growth rate variations are believed to result from the same period. Specific enzyme activities of trypsin and differences in water temperature, food availability, or other amylase indicated high values with large variance during factors (Bailey and Stehr 1986). Bailey and Stehr (1986) the early period. All three enzyme activities increased with pointed out that larvae fed with high ration levels up to 50 age after the transition period, and the specific enzyme ac- rotifers m1-1 had markedly better growth and survival. tivities became constant. The existence of two types of li- Yamashita and Bailey (1989) attempted to explain larval pase was suggested. One lipase showed a peak of specific growth from the standpoint of bioenergetics. They reported activity at Day 4 and might be related to yolk-sac absorp- the ontogenetic change of growth rate during three larval tion. The activity of the other lipase increased with age af- stages, i.e., yolk-sac period, transition period from endogter Day 14 and might be related to digestion of prey lipid. enous to exogenous food, and obligatory exogenous peOur results suggest that digestive enzymes included in food riod. The ontogenetic changes of growth rate were exorganisms supplement larval pollock digestive enzymes. plained by several energetic parameters (i.e., ingestion, egestion and metabolism), but the relation between growth rate and ontogenic changes of digestive activities or morphological development of digestive organs appeared to be Introduction of key importance. To date no information has been reported on the development of digestive mechanisms in Growth rate during the larval period is a critical factor in- walleye pollock larvae. fluencing survival rates of marine fish (Houde 1987). It is The digestive physiology of marine fish larvae is still believed that larval fish are more vulnerable to capture by incompletely understood. Many papers have been pubsome predators than juveniles because of their small size lished on the digestive physiology of fish larvae (e.g. Taand undeveloped swimming abilities; therefore, a high lar- naka et al. 1972; Lauff and Hofer 1984; Baragi and Lovell 1986; Govoni et al. 1986; Cousin et al. 1987; Ruyet et al. 1989; Segner et al. 1989), but no general relationship beCommunicatedby T. Ikeda tween ontogenetic development and digestive enzyme acY. Oozeki (~) tivities has been reported. Digestive enzyme activities of Tohoku NationalFisheries Research Institute,Japan Fisheries larval fish have been measured on pooled specimens in Agency, 3-27-5 Shinhama-cho,Shiogama,Miyagi985, Japan some studies because of the difficulty of measuring low K. M. Bailey enzyme levels. Alaska Fisheries Science Center, NationalMarine Fisheries Service, NOAA, 7600 Sand Point Way N. E., Building4, In recent years, two types of highly sensitive methods BIN C15700, Seattle, Washington98115, USA have been introduced for quantifying digestive enzymes of Abstract
178 m a r i n e fish larvae. T r y p s i n q u a n t i t y in fish l a r v a e has b e e n a s s a y e d u s i n g a radio i m m u n o a s s a y (RIA) w i t h specific a n t i b o d i e s a g a i n s t t r y p s i n p u r i f i e d f r o m the p a n c r e a s ( H j e l m e l a n d a n d J 0 r g e n s e n 1985). T h e t r y p ' s i n - R I A is sensitive e n o u g h to b e u s e d on i n d i v i d u a l larvae, a n d the secretion mechanisms of trypsin have been revealed in h e r r i n g l a r v a e ( P e d e r s e n a n d A n d e r s e n 1992). A h i g h l y s e n s i t i v e f l u o r e s c e n c e t e c h n i q u e also has b e e n u s e d to q u a n t i f y tryptic e n z y m e a c t i v i t y o f i n d i v i d u a l fish l a r v a e (Uebersch~ir 1988; Uebersch~ir et al. 1992). A l t h o u g h trypsin is an i m p o r t a n t d i g e s t i v e e n z y m e in m a r i n e fish larvae, e n z y m e s related to lipid a n d c a r b o h y d r a t e d i g e s t i o n are also i m p o r t a n t with r e s p e c t to b i o e n e r g e t i c s o f fish larvae. B u t d e t a i l e d i n f o r m a t i o n o n a m y l a s e or l i p a s e in m a r i n e fish l a r v a e is n o t a v a i l a b l e . In the p r e s e n t study w e m e a s u r e d the activities o f three d i g e s t i v e e n z y m e s , i.e., t r y p s i n - l i k e protease, lipase a n d a m y l a s e , i n d i v i d u a l l y a n d s e m i - i n d i v i d u a l l y (groups of two l a r v a e o f the s a m e s t a n d a r d l e n g t h , SL) f r o m h a t c h i n g to 39 d after h a t c h i n g in order to e x a m i n e d e v e l o p m e n t a l c h a n g e s o f d i g e s t i v e ability. E n z y m e activities o f food org a n i s m s a n d those o f the other b o d y tissues, e x c l u d i n g dig e s t i v e organs, were also m e a s u r e d a n d e v a l u a t e d .
Materials and methods Rearing of larvae Experiments were conducted from April to May 1993. Mature walleye pollock were collected by trawl in Shelikof Strait, Gulf of Alaska on 4 April 1993 by the NOAA R.V. "Miller Freeman". Eggs were hand-stripped from a single ripe female and were fertilized with sperm from several males. Eggs were allowed to incubate at ambient temperatures for 10 rain, rinsed several times in filtered seawater and placed in thermos bottles for transport to the laboratory. Temperatures on arrival at the laboratory did not exceed 5~ Eggs were incubated in the laboratory at a density of 1000 eggs per 20-liter tank until just before hatching at 5.5 to 6.0~ when they were transferred to a 120-liter black fiberglass tank containing 5 ~amfiltered seawater. Overhead fluorescent lights provided a 14-h light:10-h dark diel cycle. Water temperature was maintained between 5.2 to 6.8~ Ten to 20 percent of the seawater was exchanged each day after larvae began to hatch. Rotifers Brachionus plicatilis, cultured on Isochrysis spp., Tetraselmis spp. and Chaetoceros spp., were added to the rearing tank at a density of 1 to 5 ind ml -~ from 4 d after hatching. Rotifer density was maintained at that level throughout the rearing period. Natural zooplankton collected with 250-pm mesh plankton net and then passed through 330-pro mesh screens was fed to larvae as an additional food at naupliar densities of 0.5 to 1.0 ind ml 1 on Day 3, 15 and 18. Samples for enzyme assays were taken every day from hatching to Day 8 and every other day from Day 8 to 20. Thereafter, samples were taken on Days 23, 26, 31 and 39. Sampled larvae were anesthetized with 0.1% MS-222 in seawater, and SL (tip of snout to end of notochord) was measured to the nearest 0.01 mm with an ocular micrometer prior to freezing at -80~ We did not attempt to remove prey from larval guts before freezing or enzyme assays because of the potential loss of enzymes from guts. Samples for measuring enzyme activities of body parts other than digestive organs were taken on Days 9, 13 and 17. These samples were prepared by dissecting out digestive organs in icecooled seawater under a microscope. Eviscerated specimens were rinsed with ice-cooled seawater and then blotted. Morphological development of digestive organs was observed under a dissecting microscope on specimens preserved in 5% formalin at the same time
as enzyme activity specimens. Rotifer samples for measuring enzyme activities included in food organisms were prepared by concentrating rotifers to equal 400 to 500 ind 100 ~l-~in micro centrifuge tubes.
Digestive enzyme assays Materials were obtained from the following sources: trizma base [tris(hydroxymethyl)aminomethane], bicinchoninic acid protein assay kit, EDTA (free acid), taurocholic acid sodium salt, 4-nitrophenyl caproate, bovine serum albumin (BSA), N-~-carbobenzoxyL-arginine 7-amido-4-methylcoumarin hydrochloride (CBZ-L-ArgMCA) from Sigma; m-amylase assay kit from Boehringer-Mannheim. (Reference to trade names does not imply endorsement by the Japanese Government and the United States Government.) All other common chemicals such as NaC1 and HC1 were analytical grade. Individual frozen larvae were homogenized in 100 ~1 ice-cold homogenization buffer (20 mM tris-HC1, 1 mM EDTA, 10 mM CaC12, pH 7.5) with a Duall disposable homogenizer (1.5 ml microcentrifuge tube and a disposable tissue grinder; Kontes Glass Co., San Francisco, California) for 2 min at 130 rpm. The tip of the tissue grinder was rinsed with 300-pl homogenization buffer and the vials, containing 400 lal homogenate, were centrifuged for 30 min at 1700xg. Temperature was kept near 0~ during centrifugation. The supernatant was used for the assays of trypsin, lipase and protein content. Specimens pooled with two equal length larvae were homogenized in the same manner. The tip of the tissue grinder was rinsed with 200-gl homogenization buffer and the vials, containing 300-pl homogenate, were centrifuged as mentioned above. The supernatant was used for the assays of a-amylase, lipase and protein contents. The flourometric assay of trypsin-like enzyme activity was conducted using N-c~-carbobenzoxy-L-arginine 7-amido-4-methylcoumarin hydrochloride (CBZ-L-Arg-MCA) as a substrate. The synthesis and properties of this substrate have been described by Kanaoka et al. (1977). We adopted the modified method reported by Uebersch~ir (1988). A 50-pl supernatant of larval homogenate was added to 500-pl substrate in a cuvette and vortexed. The substrate contained 50 mM tris-HC1 (pH 8.0), 10 mM CaC12, 0.2 mM CBZ-LArg-MCA. CBZ-L-Arg-MCA was first dissolved in dimethyl-sulfoxide (DMSO) and stored at -80~ The cuvettes were left in a water bath at 30~ for 10 min and 100-pl 30% acetic acid was added for quenching (Hjelmeland and J0rgensen 1985). Blanks were prepared in the same manner except by adding t00-pl 30% acetic acid before mixing the substrate. The assays were carried out in 10x 75 mm borosilicate glass test tubes as cuvettes, and the fluorescence was measured using a Shimadzu RF-540 spectrofluorometer. The difference in emission at 440 nm (excitation 380 nm) was measured between the samples and blanks. Trypsin activities were expressed in units (U), as percentage increase of emission rain-1 following UeberschSx (1988). Activity of bilesalt-dependent lipase was determined spectrophotometrically following the method of Gjellesvik et al. (1992). Hydrolysis of 4-nitrophenyl caproate (4-NPC) was measured in an assay mixture (1 ml) containing 0.5 M tris-HC1 (pH 7.4), 6 mM sodium taurocholate and 0.1 M NaC1.4-NPC (100 mM in ethanol) was added to the assay mixture to a final concentration of 0.35 mM. A 50-91 supernatant of larval homogenate was added to the l-ml assay mixture in a cuvette and mixed. The reaction rate was measured by the increase in absorbance at 400 nm every 3 to 12 min at 30~ Reagent blanks were prepared in the same manner mentioned above by adding homogenization buffer instead of the larval homogenate at the start and end points of the measurement because of the spontaneous hydrolysis of 4-NPC. The extinction coefficient used for nitropbenol was 16300 M -1 cm ~ at pH 7.4 and 400 nm. A unit of bilesalt-dependent lipase activity (U) was defined as pmol 4-NPC hydrolyzed min-1 . c~-amylase activity was measured spectrophotometrically using an assay kit (Boehringer-Mannheim). This assay kit adopted the method of Rauscher et al. (1986). The substrate mixture included
179 105 mMHEPES buffer (pH 7.1), 52 mMNaC1, 10 mMMgCI 2, 3 mM 4,6-ethylidene-G 7 para-nitrophenol as a substrate, 24 U ml-I c~-gtucosidase (enzyme code 3.2.1.20, 25~ yeast) as an auxiliary enzyme, A total of 5 mol of substrate yield 4 tool of para-nitrophenol (PNP) by the activity of o~-amylase. A 100-~l supernatant of paired larval homogenate was added to the 1 ml substrate mixture in a cuvette and mixed. The reaction rate was measured by the increase in absorbance at 405 nm every 3 to 12 min at 30~ Blanks were not prepared, because no spontaneous increase of PNP was observed through the measurements. The extinction coefficient used for PNP was 9.9 ~tM-1 cm-I at pH 7.1 and 405 nm. A unit (U) was defined as the activity of amylase which converts 1 nmol of substrate in 1 min. Measurements of soluble protein content were carried out using the Bicinchoninic acid protein assay kit (Sigma), adapting the method reported by Smith et al. (1985). This method was thought to be more suitable for single larvae, containing a small amount of total protein, than the Lowry method (Lowry et al. 1951) or the Bradford method (Bradford 1976), because of the wide dynamic range of absorbance and the low minimum detection limit (Berges et al. 1993). Protein concentration was calculated using BSA as a standard. A 50-~1 supernatant of larval homogenate was added to the 1 ml substrate mixture and mixed. Absorbance was measured at 562 nm after incubation at 60~ for 1 h. Statistical analyses were conducted by using SYSTAT (SYSTAT, Inc., USA). Smooth line fitting of distance weighted least square method was adopted in the analyses of digestive enzyme activities by using SYGRAPH | (SYSTAT, Inc., USA; Wilkinson 1990).
Results
A
Z I
fi= 10 v =~ 8 ~ 6 ~
4 :1"
%
1'0
~ 2'0 ' Days after hatching
3'0
40
10
20 Days after hatching
30
40
B
300 ~ a, 200 .E ~ 100 ao
o
300
Larval growth in SL was not linear (Fig. 1A) so it appeared suitable to divide development into three growth periods following Yamashita and Bailey (1989). The day of 50% hatching was designated as the day of hatching o f all larvae. The upper and lower jaws were developed on Day 3 and the mouth opened on Day 4. Food organisms were observed in the guts of most larvae after Day 5. Therefore, Day 5 was designated as the onset of feeding. Average live SL of newly hatched larvae was 4.93 mm, and the protein content was 23.3 btg. On Day 5 (onset of feeding), larvae averaged 5.82 m m SL and 15.9 pg protein. From morphological observation, yolk was mostly absorbed by Day 7 and completely absorbed by Day 20 (Fig. 2). The average 20-d old larvae was 6.90 m m SL and 47.9 g g protein. After Day 20, the variance between individuals increased; larvae averaged 8.98 m m SL and 124.5 pg protein on Day 39. The linearized growth rates in SL were 0.19 m m d q during yolk-sac period (from Days 0 to 5), decreasing to 0.05 m m d -I during the transition from endogenous to exogenous energy (from Days 5 to 20). Afterwards growth rate increased to 0.12 m m d 1 up to Day 39. Growth rate in protein content from Days 0 to 5 was negative at -1.28 Pg d q and then increased to 1.53 ~g d q during Day 5 to 20 (Fig. 1B). The growth rate in protein content increased to 6.91 pg d -1 after Day 20. The relationship between the SL (in mm) and the protein content (P, in ~g) was expressed by the following two regression equations (Fig. 1C): Days 0 to 5: P = 5 1 . 2 - 5 . 7 7 S L (r=0.567, N = 2 4 , p 40 "5 .E .m
~,
2o
0
m
4
5
0.6 D
9
9
6 7 8 9 10 Standard length (mm)
11
12
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s ~0.4 O3
II
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~5 0.2 c 69 Q.
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o
I
4
5
6
9 1~0 Standard length (mm) 7
8
,
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11
12
Fig. 3 Theragra chalcogramma. Relationships between A total trypsin activity and age (in d), B specific trypsin activity and age (in d), C total trypsin activity and standard length, D specific trypsin activity and standard length. Values are means +SD of three specimens in A and B. Smooth fitting lines were drawn by distance weighted least square method using SYGRAPH|
182 1.2
A
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.,
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