Chitinolytic enzymes in the integument and midgut-gland of the shrimp Palaemon serratus during the moulting cycle. M. Spindler-Barth 1, A. Van Wormhoudt 2 ...
Marine Biology106, 49-52 (1990)
Marine BiOlOgy
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© Springer-Verlag 1090
Chitinolytic enzymes in the integument and midgut-gland of the shrimp Palaemon serratus during the moulting cycle M. Spindler-Barth 1, A. Van Wormhoudt 2 and K.-D. Spindler 3 1 Institut ffir Zoologie,Abt. Parasitologie, UniversitfitDfisseldorf,Universit/itsstraBe 1, D-4000 Diisseldorf, FRG 2 Laboratoire de Biologie Marine, Coll~gede France, F-29181 Concarneau, France 3 Institut ftir Zoologie, Lehrstuhl fiir Hormon- und Entwicklungsphysiologie,Universit/it Diisseldorf, UniversitfitsstraBe1, D-4000 Dtisseldorf, FRG Date of final manuscript acceptance: April 18, 1990. Communicatedby O. Kinne, Oldendorf/Luhe
Abstract. Chitinase and N-acetyl-/~-D-glucosaminidase activity were quantified in Palaemon serratus (Pennant) integument and midgut gland during the moulting cycle. Studies were performed on specimens collected near Concarneau, France, in July 1989. The changes in specific activity are different in the two organs: in the midgut gland enzymatic activity is high throughout the whole moulting cycle with a weak peak at the early premoult Stage D 1', whereas in the integument the activity of both enzymes is very low throughout post- and intermoult stages and rises only after premoult Stage DI'. The highest specific activity is reached in DI'" for chitinase and somewhat earlier (DI") for N-acetyl-/%D-glucosaminidase. The increase in specific chitinolytic activity coincides with an increase in ecdysteroids.
Introduction Chitin is a polymer of fl (l-4)-linked amino-sugars, predominantly N-acetylglucosamine and to a lesser degree also glucosamine. It is distributed world-wide in a variety of plant and animal phyla and represents the carbohydrate with the second largest biomass. It is degraded by exo- and endochitinases (Spindler 1983, Kramer etal. 1985, Chen 1987). These enzymes are not only involved in resorption of parts of the old cuticle during the moulting cycle but they also function as hatching enzymes e.g. in Artemia salina (Funke and Spindler 1987, Funke et al. 1989) and as digestive enzymes. This was demonstrated in a variety of species, for example in different crustaceans (Arnould and Jeuniaux 1982). The simultaneous presence of chitin degrading enzymes in the integument and the digestive tract, and their different functions in these organs, raises the question whether moulting hormones (ecdysteroids) are only involved in the regulation of chitinolytic activity in integument or whether they also influence those chitin degrading enzymes which are involved in digestion of food but not in apolysis. The first indication that changes in chitinolytic activities in crustacean integument was related to
moulting was presented by Jeuniaux (1963), but a more thorough investigation including all moulting stages has only been studied in insects (for reviews see Spindler 1983, Kramer et al. 1985, Spindler-Barth et al. 1986). Recently however, comparable investigations on chitin degrading enzymes in crustaceans have been performed, using the brine shrimp Artemia salina (Funke and Spindler 1987, 1989, Funke et al. 1989) and two krill species Euphausia superba and Meganyctiphanes norvegica (Spindler and Buchholz 1988, Buchholz 1989). Even less is known on the regulation of the titer of those chitinolytic enzymes which are involved in digestion. A comparison of the titer of chitin degrading enzymes in integument and digestive tract has been studied so far only in the insects Bombyx mori (Kimura 1977) and Locusta migratoria (Sommer and Spindler unpublished results) and only very recently in the krill Euphausia superba (Buchholz 1989), where a different pattern of enzymatic activity was found in the two organs during the moulting cycle, with rather high activities in the digestive tract also at the time of exuviation. This was interpreted as an "adaptation to the continuously high demand for food utilization". The present study was carried out on Palaemon serratus, a temperate species with different nutritional and energetic demands, to investigate the regulation and concentration of chitinolytic enzymes in integument and midgut gland. Materials and methods Specimens Live Palaemon serratus (Pennant) were purchased from a commercial fishermanin July 1989 and kept in the laboratory in tanks at ambient temperature (18° to 20 °C) in running seawater in natural light conditions. Moult staging and sample preparation Moult stages were determined according to Drach and Tchernigovtzeff(1967). Freshlyexcisedmidgut-glandand integument(only
50 from the abdomen) from a single specimen were homogenized in 1 ml of a 0.2 M Na-citrate-phosphate buffer, pH 5.5, with ultrasonic power (microtip) at 4°C. After centrifugation (10000xg, 5 rain 4 °C) the supernatant was used for the enzyme assays and for protein determination according to Bradford (1976), using bovine serum albumine as a standard.
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The formation ofp-nitrophenol from p-nitrophenyl-N-acetyl-/~-Dglucosaminide was taken as a quantitative indicator for N-acetyl-/~D-glucosaminidase activity as previously described (Spindler 1976). Blanks containing no enzyme were always run in parallel. The chitinase assay was performed as follows: 3H-labelled chitin was prepared according to Molano et al. (1977) and used as substrate. An aliquot (40 #1) of the 3H-chitin-suspension (12.5 mg m1-1) was mixed with 5 to 50 #1 of the homogenate and 0.2 M citrate-phosphate buffer, pH 5.5 added up to 150 #1. This mixture was incubated for 5 h under continuous stirring at 35 °C. The reaction was stopped by adding 200 #1 of ice-cold 10% trichloro-acetic acid and centrifuged for 5 min at 10000 x g to remove the non-degraded chitin. N-acetyl-/%D-glueosamine and its oligomeres are the endproducts of the reaction. They remain in the supernatant. An aliquot (200/~1) of this supernatant was mixed with 3 ml of scintillation cocktail and the radioactivity determined .with a liquid scintillation counter (Tricarb 460C, Packard). Controls without extract were treated in the same way and substracted from the experimental values.
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Chitinolytic activities in the integument The titer curves for the specific activities of chitinase (Fig. 1 a) and N-acetyl-/%D-glucosaminidase (Fig. 1 b) in Palaemon serratus are different. Both enzymes have a rather low specific activity during post- and intermoult. The activity increases beginning with the pre-moult Stage D I ' and reaches the highest values in DI" for the N-acetyl-/~-D-glucosaminidase and in D I ' " for the chitinase. The increase in chitinase activity in the moulting Stage D I " is significant when compared to the preceeding moulting stages (Students' t-test = 7.1 ; f = 4; p < 0.01 ) and this also holds true if one compares the value from the peak m a x i m u m with the post- and intermoult and the latest premoult stages. Chitinase activity decreases shortly before exuviation, whereas a second peak of activity for the N-acetyl-/%D-glucosaminidase occurs. Both maxima of the N-acetyl-/~-D-glucosaminidase are significant (p < 0.05) when compared to the values during post-, intermoult and early premoult stages. The increase in activity is a b o u t 20-fold for the chitinase and eight-fold for the N-acetyl-/~-D-glucosaminidase. Chitinolytic activities in the midgut gland In contrast to the integument, both enzymatic activities show less pronounced changes during the moulting cycle in the midgut gland (Fig. 2). T h r o u g h o u t all moulting stages there is a rather high specific activity with an approximately two-fold increase at the premoult Stage D I ' for both enzymes. The increase in N-acetyl-/%D-
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Fig. I. Pa/aemon serratus. Specific chitinase (a) and N-aeetyl-/~-Dglucosaminidase (b) activity in the integument of shrimp during the moulting cycle. Each point represents the mean of 4 to 6 individuals. Standard deviations are less than 10%, except the peak maxima (20 to 30%)
glucosaminidase-activity is significant (Students' t-test = 2 . 6 6 ; f = 11; p < 0.05) as compared to the lowest values in the Stages C or D3, which is not the case for chitinase ( f = 4; p > 0.05). Even at the stages with the lowest values the absolute activities are much higher compared with maximal activity in the integument.
Discussion
The titer curves of chitinase and N-acetyl-/?-D-glucosaminidase in integument of Palaemon serratus resemble those found in insects and the few crustaceans investigated so far. F o r example, an earlier increase in N-acetyl/%D-glucosaminidase as well as a double-peaked curve as compared to chitinase has also been described in insects (Zielkowski and Spindler 1978, Spindler-Barth etal. 1986, Fukamizo and K r a m e r 1987) and also in Euphausia superba (Buchholz 1989). The pronounced decrease in chitinase activity just before exuviation is c o m m o n to all insects and also to Artemia salina (Funke et al. 1989) but was not found in E. superba (Buchholz 1989) which m a y
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2. Palaemon serratus. Specificchitinase (a) and N-acetyl-/%Dglucosaminidase (b) activityin the midgut gland of shrimp during the moultingcycle.Each point represents the mean of 4 to 6 individuals. Standard deviationsare less than 10%, except the peak maxima (20 to 30%) Fig.
be due to a problem of staging at the far end of the moulting cycle in this species (Buchholz 1989). In Maja squinado a rapid increase in chitinase activity after Stage DI' has also been described, reaching two-and-a-half to six-fold higher values in the peak maxima as compared to Stages C and DI' (Jeuniaux 1960). An approximately six-fold increase in specific activity also occurs in PaIaemon serratus (see Fig. 1 b). The difference in the pattern of chitinase activity throughout the moulting cycle between M. squinado (constant) and P. serratus (pronounced peak at Stage DI"') can easily be explained, since Jeuniaux (1960) only studied epidermis whereas we used complete integument, which contains the exuvial fluid rich in chitinase at later premoult stages. The increase in chitinolytic activities in P. serratus in Stage D1 coincides with the increase in moulting hormones in the same species (Baldaia et al. 1984, Van Wormhoudt et al. 1985). This close relationship between the titer of moulting hormones and chitinolytic enzymes is common to all arthropods so far investigated under this aspect (Spindler 1983, Baler and Scheffel 1984, Spindler-Barth et al. 1986,
51 Chen 1987, Funke and Spindler 1987, Buchholz 1989, Funke et al. 1989), although direct stimulation of chitinolysis by moulting hormones has been described only in the insects Manduca sexta (Fukamizo and Kramer 1987) and Bombyx mori (Kimura 1973) and in the barnacle Balanus amphitrite (Freeman and Costlow 1979, Freeman 1980). Our results on the chitinolytic activities in the midgut gland can be compared with those from the antarctic krill Euphausia superba (Buchholz 1989) and from Maja squinado (Jeuniaux 1960). There are several features in common: (1) The activities of both enzymes are higher in the midgut gland than in the integument which holds true even if one compares the minimal values in midgut gland with the peak values in the integument. (2) There is a different pattern of enzymatic activity during the moulting cycle in midgut gland as compared to the integument. (3) The activities are high throughout the whole moulting cycle even around exuviation. (4) The differences between minimal and maximal values are only two-fold or even less. In Palaemon serratus there is a weakly pronounced peak at the premoult Stage DI' both in chitinase and N-acetyl-/?-D-glucoseaminidase activity in the midgut gland. This peak of activity does not coincide with the peaks of the corresponding enzymes in the integument of the same species and is also different from the titer of another digestive carbohydrase of this species, namely amylase, which shows peaks at the moulting Stages B and DI'" (Van Wormhoudt 1983). Obviously the digestive enzymes of the midgut gland of P. serratus, including chitinase and N-acetyl-//-D-glucosaminidase, are under a different control mechanism than the corresponding enzymes in the integument. One explanation for this difference could be that the two organs possess different isoenzymes, some of which could be constitutive and therefore expressed throughout the whole moulting cycle such as the midgut gland enzymes and others in the integument could be induced by moulting hormones. An induction of an N-acetyl-~-D-glucosaminidase by a steroid hormone has been shown in chick oviduct (Lucas 1979). Further experiments have to clarify whether the isoenzyme pattern in the two organs is different and whether ecdysteroids are able to induce chitinolytic activity in the integument. Acknowledgement. We greatfullyacknowledgethe correction of our English by Dr C. Bridges (Diisseldorf). Literature cited
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