Chitinolytic enzymes in the digestive system of marine fishes

Chitinase, exo-N-acetyl--D-glycosaminidase (NAGase) and lysozyme activities were assayed in the digestive tract of 6 species of marine fishes: Myxine glutinosa (cyclostome), Chimaera monstrosa (holocephalan), Squalus acanthias, Etmopterus spinax, Raja radiata (elasmobranchs) and Coryphaenoides rupestris (teleost). Strong chitinase activity was found in the gastric mucosa of the elasmobranchs (S. acanthias, E. spinax and R. radiata) and the teleost (Coryphaenoides rupestris). A remarkably high chitinase activity occurred in the pancreas of the stomachless holocephalan fish Chimaera monstrosa. NAGase activity was strong in the digestive tract of all species. It could be concluded that marine fishes with diets consisting largely of chitinous invertebrates may display high chitinase and NAGase activities in their digestive system; however, only low chitinase activity was found in the intestine of the cyclostome Myxine glutinosa. Coryphaenoides rupestris gastric mucosa chitinase had one optimum activity at pH 1.25, whereas S. acanthias chitinase had two optima, at pH 1.6 and 3.6. The NAGase pH-activity curves from S. acanthias and R. radiata gastric mucosa displayed similar optima, at pH 4.5 and 4.25 respectively. Chimaera monstrosa pancreatic chitinase had a very strong optimum around pH 8 to 10, and one less strong at pH 3. These enzyme activities could not be separated by gel filtration or isoelectric focusing. The pI (isoelectric point) was approximately 4.9 for both enzymes. The molecular weight of the C. monstrosa pancreatic chitinase was estimated to be approximately 43 000. Lysozyme activity was absent or extremely weak in the material studied.     

Lipid-class composition of organs and tissues of the tiger prawnPanaeus esculentus during the moulting cycle and during starvation

Neutral lipid and phospholipid fractions and their component lipid classes in the digestive gland, abdominal muscle, epidermis and cuticle ofPenaeus esculentus Haswell were analysed and compared during the moulting cycle and during starvation. The prawns were collected from Moreton Bay, Queensland, Australia, by trawling during 1985–1987, and were fed on a standard, semi-purified diet. The digestive gland appears to be a major site of lipid synthesis, storage and mobilisation in preparation for moulting. Neutral lipid, 59 to 80% of which was triacylglycerol, was the larger fraction. It accumulated during early premoult, mainly due to the increase in triacylglycerol. The digestive gland contained only 18% of the total body lipid, or 8% of body lipid as triacylglycerol. Thus, the reserve lipid available for energy production is very small. Digestive gland triacylglycerol was markedly depleted after 4 d starvation and was almost completely absent after 8 d. In the other tissues, the major fraction was phospholipid, of which over 50% was phosphatidylcholine and up to 20% phoshatidylethanolamine; cholesterol was the major class in the neutral lipid fraction and appeared to be very stable. Most of this lipid was probably a component of cellular membranes. The lipid composition of muscle changed very little during the moulting cycle: total lipid levels in the epidermis were high in late premoult and early postmoult, when new cuticle is being secreted, but the proportions of the component lipids were closely similar. Cuticle lipid, together with other major components, was resorbed from the old cuticle prior to ecdysis, but the cuticle phospholipids appeared to be labile at all moult stages. The total of all lipids in fedP. esculentus was about 3.6% dry weight, of which about 70% was phospholipid. Earlier research had shown that when digestive gland lipid is exhausted after a short period of starvation, muscle is metabolised for energy. The present research showed that in the remaining muscle only about 13% of lipid was lost after 21 d starvation, mostly as phosphatidylcholine. This is in keeping with the need to maintain this tissue in a functional state. In contrast, epidermal lipid levels were markedly reduced after only 4 d starvation and the proportions of phospholipids changed significantly. This sensitivity of the cuticle lipids to starvation may be the cause of delayed moulting, which is characteristic of poor nutrition.     

Fatty acid composition of organs and tissues of the tiger prawn Penaeus esculentus during the moulting cycle and during starvation

The fatty acids (FA) in neutral lipid (NL) and polar lipid (PL) of digestive gland, muscle and integument of Penaeus esculentus Haswell were analysed and compared during the moulting cycle and during starvation. The prawns were collected from Moreton Bay, Queensland, Australia, by trawling during 1985–1987, and were fed with a standard semi-purified diet. Compared with a natural diet, the artificial diet had much higher levels of 18:1n-9 and 18:2n-6, but only trace amounts of 20:4n-6, but there was no evidence of dietary imbalance. The fatty acid composition (percentage of total lipid) of the digestive gland changed markedly during the moulting cycle and during starvation, but the small changes observed in both muscle and integument suggested that these tissues maintained their composition under both conditions. When the fatty acids were calculated as absolute amounts, muscle composition, as well as that of the digestive gland, changed significantly. In the digestive gland, saturated FA (SFA), monounsaturated FA (MUFA), diunsaturated FA (DUFA) and polyunsaturated FA (PUFA) all increased up to the middle of the moulting cycle and then declined; with starvation all groups decreased. In muscle, SFA, MUFA and DUFA all increased during the moulting cycle; starvation caused SFA, MUFA and PUFA to decrease, whereas DUFA did not vary. Starvation caused both 18:2n-6 (linoleic) and 18:3n-3 (linolenic) in the digestive gland to reach or almost reach zero. The other essential PUFA, 20:4n-6 (arachidonic), 20:5n-3 (eicosapentaenoic) and 22:6n-3 (docosahexaenoic), decreased during the moulting cycle, but during starvation 20:4n-6 did not decrease as much. In muscle, the levels of 18:2n-6 and 18:3n-3 increased, while 20:4n-6, 20:5n-3 and 22:6n-3 remained approximately constant during the moulting cycle. Starvation reduced 20:5n-3 and 22:6n-3 to about 60%. The data suggest that levels of 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3 are regulated, and that 20:4n-6 can be synthesised from 18:2n-6. There is no clear evidence that 20:5n-3 and 22:6n-3 are essential in P. esculentus, but tissue catabolism of cell membranes during starvation may have provided sufficient amounts for maintenance.     

Blood sugar metabolism during moulting in the isopod crustacean Ligia exotica

Blood sugar metabolism of the isopod Ligia exotica Roux was studied in relation to chitin synthesis during the moult cycle. The blood sugars were found to occur free, as well as bound with blood proteins, during all moult stages, as in Emerita asiatica. The variety of free blood sugars is smaller in L. exotica; it comprises only glucose and glucose-6-phosphate. The protein-bound blood sugars are glucose and glucosamine. The blood-volume values do not differ markedly during the moult cycle. Estimations of blood sugars showed a marked rise of sugar levels in the blood during the premoult phase, and a subsequent fall during the freshmoult phase, when chitin is actively synthesised. The chitin values also showed a definite correlation with the blood-sugar values. The increased resorption of chitin during the premoult stage, and its liberation into the blood stream as glucosamine, is indicative of its reutilisation in chitin synthesis as in E. asiatica and the genus Cecropia. As more immediate precursors of chitin, such as uridine diphosphate and uridine diphophate acetylglucosamine compounds, were absent in the blood, a thorough biochemical study of the epidermis is suggested to locate the intermediate precursors of chitin which are possibly derived from blood sugars.     

Haemocyanin concentration of juvenile lobsters (Homarus gammarus) in relation to moulting cycle and feeding conditions

Groups of laboratory-reared, juvenile lobsters (Homarus gammarus, 40–50 g wet weight) were given different types of food (shrimp, mixed invertebrates and pellets, bivalves, starved) for a period of 22 wk. Haemocyanin concentration was measured weekly, moult stage and moultings noted. Haemocyanin concentration decreased rapidly in the starved group (from 0.8–0.6 to 0.2 mM). The group fed with bivalves showed a small decline with time, to 0.5–0.3 mM at the end of the period, indicating a deficient diet. The haemocyanin in the other groups did not change significantly during the experiment. Moultings decreased in frequency and the intermoult period was prolonged when the quality of food was decreased, and in the starved group moulting ceased completely after 1.5 months. Haemocyanin concentration was lowest immediately after moult and increased gradually through stages C and D. Lobsters that were starved or maintained on the bivalve diet did not reach the same premoult haemocyanin levels as those fed with shrimps and mixed food.     

Ultrastructure of the integument of a pelagic Crustacean: moult cycle related studies on the Antarctic krill,Euphausia superba

The ultrastructure of euphausiid integument was examined in relation to the moult cycle and supplemented by investigations of chitinase activity in the integument and content of N-acetyl--D-glucosamine in the hemolymph. The Antarctic krill, Euphausia superba was collected in 1983 in Admiralty Bay, King George Island, Antarctica. Some specimens of the Northern krill, Meganyctiphanes norvegica, from the Danish Kattegat served for comparison. As a major aim of the study, the moult staging system developed for living tissue could be verified by ultrastructural findings. Under experimental high production conditions of the Antarctic summer, no period of rest or intermoult between post- and premoult was observed in subadult E. superba. Neither was a resting phase seen at the cellular level, the epidermis remained active. The epidermal gland cells did not show any cyclical changes, and the organelles of protein synthesis were generally well developed in all moult stages. In order to follow the physiological course of events, structural and biochemical methods were combined and showed as a result that the last moult stage before ecdysis is characterized by massive cuticular resorption. The epicuticle remained ultrastructurally unchanged before and after ecdysis, even though its permeability should alter at ecdysis. The existence of muscle insertions which connect the old and the new cuticle across the exuvial space suggests an answer to the question why E. superba is hardly impaired in swimming almost up to the time of ecdysis.Dedicated to Professor Dr. H. Leonhardt on the occasion of his 70th birthday     

Breeding and moulting of barnacles under rearing conditions

The reproductive parameters of three species of barnacles common in warm-water regions,Balanus amphitrite Darwin,B. eburneus Gould andB. trigonus Darwin, cultured singly or in pairs for 2 1/2 yr, were compared. All specimens, collected from Tokyo Bay and studied between 1985 and 1987, were fed on newly hatchedArtemia sp. larvae (Tetra strain) and phytoplankton culture and were maintained under constant temperature (20°C) and controlled photoperiods (10 h light:14 h dark). The breeding frequencies of these three species were equally high, from 2 to 4 broods mo^–1 in the first year; they were relatively high between October and February, averaging 3 broods mo^–1.B. amphitrite, B. eburneus andB. trigonus produced 24, 21 and 11 broods yr^–1, respectively. Brood size was quite variable among the three species examined, yielding 4000 embryos brood^–1 inB. amphitrite andB. eburneus and 6000 inB. trigonus. Brood interval averaged 4 to 9 d. The moult occurred over short intervals of 4 to 10 d moult^–1. Both breeding and moulting frequencies were influenced by low temperature and starvation. Self-fertilization was observed in all species and was more frequent inB. amphitrite than inB. eburneus andB. trigonus. Nutritional condition and temperature seemed to be the main external factors regulating both breeding and moulting processes in barnacles.     

Long-Term observations on the moulting frequency of the shrimp Lysmata seticaudata

The effects of body-size and temperature on moulting frequency of the shrimp Lysmata seticaudata Risso were examined under controlled laboratory conditions. Shrimp ranging in weight from approximately 0.4 to 1.14 g and maintained at 17°C exhibited intermoult periods of similar duration (20 d). On the other hand, small individuals (0.23 g) held at the same temperature moulted on the average every 14.5 d. Over a temperature range from 8° to 20°C mean intermoult periods were inversely and linearly related to temperature; an increase of 1 C⁰ resulted in a decrease in the intermoult period of approximately 1.5 d. Although temperature acted to regulate the intermoult period of each shrimp in increments of whole days, it had little effect on the time of moulting within any 24 h period, since moulting occurred more than 93% of the time between 18.00 and 08.00 hrs. The night-time moulting pattern also tended to be most pronounced at lower temperatures (8° and 13°C) and in larger individuals. Possible factors controlling this phenomenon and its occurrence in other crustacean species are discussed.     

木薯皮堆肥过程中酶活性的变化

以木薯皮为原料进行高温堆肥发酵,研究了堆肥化过程中理化性质和酶活性的变化情况.结果表明:在堆肥发酵过程中.温度变化呈先上升后下降趋势,到后期已趋近于初始温度;而含水率在堆肥处理过程中变化不大.各处理的pH值在发酵结束时为7.3～7.5.过氧化氢酶活性初期较高,随后迅速降低,并维持在较低水平.纤维素酶和脲酶活性在堆肥初期增加,之后逐渐降低.由于堆肥原料不同,不同处理的酶活性变化趋势表现出一定的差异.考虑到堆肥腐熟度受多方面因素的影响,判断堆肥腐熟度时应根据多种指标(包括生物学、化学、物理学指标)综合判断.     

Hydrobiological parameters during an annual cycle in the Arcachon Basin

Temperature, salinity, nutrients and phytoplankton biomass were monitored on a weekly to bimonthly frequency at six stations in the Bay of Arcachon from July 1984 to July 1985. This particular period appears to have differed from the last ten years in displaying higher amplitudes of both temperature and salinity (1.5° to 24.5°C and 20 to 34.5) at high tide. However, although in spring both temperature and salinity were normal (14°C, 28), an important phytoplankton spring bloom occurred, with maximum chlorophyll levels reaching 15 g l^-1 in April. Utilization of nutrients was high, particularly for nitrate and silicate, the concentrations of which decreased, respectively, from 10–15 to 0.1–2 M and 10–20 to 0.25–3 M from February to May. Exhaustion of nitrate was observed in May, except in areas subjected to river input. In contrast, silicate increased throughout the study area from May to July.     

Hydrolytic enzymes in the coelomic cells of the polychaeteNereis virens during sexual maturation

Nereis virens were collected between April 1989 and April 1990 at Yerseke, Oosterscheldt Bay, The Netherlands. Activities of the hydrolytic enzymes arylsulfatase, acid phosphatase, N-acetylglucosaminidase, aminopeptidase, and carboxylesterase were investigated in coelomic cells (elaeocytes) of individuals at different stages of maturation as determined by the time course of oocyte growth. On a protein-content basis, up to ten-fold higher specific activities were present in the elaeocytes compared to the body-wall tissue. Acid phosphatase, N-acetylglucosaminidase, leucine aminopeptidase, and long- and medium-chain esterase increased continuously from the beginning to the later stage of sexual maturation, with a decline in prespawning individuals. Shortchain esterase and acid aminopeptidase did not show corresponding changes. Activities at different maturation stages were similar for males and females, with the exception of arylsulfatase activity, which was sex-specific and almost lacking in male elaeocytes. The high hydrolytic activities present in the elaeocytes as well as the increase in these activities during sexual maturation are in agreement with the function of these cells in phagocytizing muscle fragments (sarcolytes), which arise as a result of drastic histolysis of the body musculature ofN. virens during the process of sexual maturation.     

On the timing of moulting processes in reproductively active Northern krill Meganyctiphanes norvegica

The interactions between moult phasing, growth and environmental cues in Northern krill (Meganyctiphanes norvegica) were examined through analysing populations at seasonal, weekly, and daily timescales. The analyses were carried out on resident populations of krill found in three different neritic locations that experience similar environmental signals (the Clyde Sea, Scotland; the Kattegat, Denmark; Gullmarsfjord, Sweden). Seasonal analyses were carried out on the Clyde Sea population and showed that moulting frequency increased significantly moving from winter to summer. The proportion of moulting females in summer samples was often more than double the proportion of moulting males, suggesting that females had a comparatively shorter intermoult period (IMP). Weekly samples taken from the Kattegat showed a similar pattern. However, although the difference between the proportion of female and male moulters was significant in one week, it was not another, mainly because of the variability in the proportion of female moulters. Such variability in females was equally evident in the daily samples taken at Gullmarsfjord. It suggests that females have a shorter IMP (12.5 days) than males (18.4 days) and are more likely to moult in synchrony. Nevertheless, the daily samples revealed that males are also capable of moult synchronisation, although less frequently than females. Shortened IMPs in females were not a result of the abbreviation of specific moult stages. Accordingly, reproductive activity did not alter the course of the normal moult cycle. There was no significant difference between the total body lengths of males and females indicating that females achieve the same levels of growth despite moulting more frequently and having to provision the energy-rich ovaries. This is in contrast to most other crustaceans where the energy costs of reproduction reduce female growth. The fact that females were less abundant than males, probably by suffering a greater level of mortality, suggests that different behavioural strategies, particularly vertical migration regimes, were adopted by each sex to maximise growth and reproduction.     

Variation in lipid classes during the molting cycle of the prawn Penaeus japonicus

The variation in the concentration and fatty acid composition of lipid classes during the molting cycle of the prawn Penaeus japonicus was investigated. The lipid concentration of the whole body reached a maximum at mid-premolt (Stage D₂) and then decreased to low level at late premolt (Stage D_3–4). The accumulation of lipids during the premolt period seemed to be attributable to the increase of both polar and neutral lipids. The increase of neutral lipids at Stage D₂ was derived from not only triglycerides but also free sterols and free fatty acids. Regarding the fatty acid composition of every lipid class, a marked variation occurred mainly at the intermolt (Stage C). In this stage, the polar lipids were rich in monoenoic acids such as 18:1 and poor in polyenoic acids such as 20:53 and 22:63. The triglycerides were rich in polyenoic acids at Stage C, but poor in monoenoic acids such as 16:1 and 18:1. The steryl esters contained large amounts of saturated acids such as 16:0 and 18:0 throughout the molting cycle, however the level of polyenoic acids increased at Stage C.     

Ultrastructure and histochemistry of the integument of air-breathing polychaetes from mangrove swamps of sumatra

The integument of S.E. Asian air-breathing polychaetes exhibits a variable ultrastructure with regard to cuticle and epithelial cells. The cuticle varies in thickness, contents of regularly arranged collagen fibrils, and structure of the epicuticle. The basal portion of the epithelium is either of smooth outline, or consists of slender processes of the epidermal cells separated by wide intercellular spaces which are invaded by a loose system of glia cells and nerve fibres. Extracellular spaces within the epidermis of the gills of Dendronereides heteropoda are transformed into blood lacunae. The enzyme content of the epithelial cells is relatively low.Supported by the Deutsche Forschungsgemeinschaft (Sto 75/2, We 380/4).     

Growth and moulting of Neomysis integer (Crustacea: Mysidacea)

The growth and moulting of Neomysis integer (Leach) was investigated in the field and the laboratory. In the Ythan estuary, Aberdeenshire, Scotland, monthly samples taken from November 1976 to October 1978 revealed that the summer generation juveniles and mature individuals grew at a rate of 4 to 5 mm and 1 to 2 mm monthly, respectively. The winter generation had a growth rate of 3 to 4 mm monthly for juveniles and about 1 mm for mature individuals; during the winter there was a period of 3 mo when growth was almost completely stopped. Mysids reared in the laboratory on Artemia sp. nauplii had an average daily growth rate of 0.06 mm at 9°C and 0.09 mm at 16°C. The growth factors of N. integer ranged from 3 to 17% for mature and immature individuals, respectively. Intermoult periods ranged from 3 to 7 d in immature mysids to 12 to 18 d in mature mysids. Average laboratory growth curves calculated from information on growth factors and intermoult periods indicate that at 9°C (winter generation) it takes N. integer 277 d to grow to be a 15 mm mature individual, whereas at 16°C (summer generation) it takes 188 d. N. integer moults 24 times as it grows from a juvenile to a mature individual.     

Chitobiase activity in the epidermis and hepatopancreas of the fiddler crab Uca pugilator during the molting cycle

The activity of chitobiase, also known as N-acetyl-β-glucosaminidase, in the epidermis and hepatopancreas of the fiddler crab Uca pugilator (Bosc, 1802), during the molting cycle, was investigated. A pH optimum of 5 to 6 was found for the enzymatic activity in both the epidermis and hepatopancreas. The temperature optimum for epidermal and hepatopancreatic chitobiase activities was 50 to 60 °C. The K _m values for epidermal and hepatopancreatic chitobiase activities at 19 °C were 0.190 ± 0.027 and 0.203 ± 0.016 mM 4-methylumbelliferyl-N-acetyl-β-glucosaminide, respectively. Hepatopancreatic chitobiase activity was significantly higher than epidermal enzymatic activity in all the molt cycle stages tested except Postmolt Stage A-B. Chitobiase activity varied significantly during the molting cycle, with the epidermal enzymatic activity in Premolt Stage D_3–4 significantly higher than in Stage C (intermolt) and Premolt Stage D₀, whereas hepatopancreatic chitobiase activity in Premolt Stage D_3–4 was significantly higher than in all other molt stages tested. The patterns of chitobiase activity in the epidermis and hepatopancreas correlate well with the hemolymph titer of ecdysteroids in U. pugilator during the molting cycle; this suggests that chitobiase activity in both tissues is regulated at least in part by the steroid molting hormones. Received: 6 May 1998 / Accepted: 12 September 1998     

Analysis of blood chemistry and flow cytometry of gonadal cell cycle during reproductive cycle of Anabas testudineus

The population of climbing perch, Anabas testudineus, an important food fish in India, is presently under threat and has been declared endangered. A narrow range of environmental conditions for successful reproduction of the species was previously ascertained in breeding experiments. In this study, the role of physiological parameters including plasma glucose, total protein, cholesterol, and glycogen content in liver and ovary were determined with respect to the reproductive cycling stages. Flow cytometric analysis of cell cycle of ovary and testis in relation to reproductive cycle was also analyzed. In both the sexes, plasma glucose and plasma protein changed significantly during breeding cycle. Plasma cholesterol levels were reduced markedly during spawning. Liver and ovarian glycogen levels showed significant variation during reproductive cycling. Flow cytometric study revealed that the hypoploid and synthesis phase were altered significantly with breeding cycle in ovarian and testicular cells. Data indicate that the biochemical parameters indicative of the fish reproduction capacity are influenced by environmental conditions. Thus, climatic changes may therefore potentially induce biochemical alterations that may exert detrimental effects on fish reproduction and subsequent population decline.     

汕头海域几丁质酶产生菌的筛选及其几丁质酶编码基因研究

在汕头海域表层沉积物中分离得到69株高产几丁质酶的菌株,对其中6株形态特征差异比较明显、几丁质酶活力比较高的菌株SWCH-1、SWCH-2、SWCH-3、SWCH-5、SWCH-6和SWCH-9进行了16S rDNA序列鉴定,发现它们分别归属于5个属.即短芽孢杆菌属(Brevibacillus)、类芽孢杆菌属(Paenibacillus)、芽孢杆菌属(Bacillus)、气单胞菌属(Aeromonas)和寡养单胞菌属(Stenotrophomonas).采用兼并引物对6株菌几丁质酶编码基因的催化保守区片段进行了PCR扩增和序列分析,发现菌株均含有18家族几丁质酶编码基因;但是其编码的蛋白序列与NCBI收录的蛋白序列存在着差异,其中菌株SWCH-1和SWCH-3的蛋白序列与数据库中序列的相似性比较低,分别为85%和81%,菌株含有比较新的几丁质酶编码基因,其全基因序列的克隆和酶蛋白的纯化分析尚在进一步研究中.图6参18     

Effects of non-circadian light/dark cycles on the growth and moulting of Palaemon elegans reared in the laboratory

The growth and moulting of Palaemon elegans Rathke has been compared under a circadian (12 h light: 12 h dark) and two non-circadian (8 h light:8 h dark and random light:dark) light/dark cycles. In prawns reared individually from hatching to the late juvenile phase, growth, measured as increase in total length, was significantly retarded in the non-circadian regimes, during zoeal, post-larval and early juvenile development. This effect was primarily the result of reduced increments at moulting in the non-circadian regimes. Growth of prawns reared from hatching to the post-larval phase, measured as wet and dry weights, was similarly reduced in a non-circadian regime. These effects support the hypothesis that the integrity of an animal's physiology is partially dependent on maintaining, through the action of daily environmental cycles, correct timing relationships between its oscillating sub-systems.