Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated <Emphasis Type="Italic">Octopus maya</Emphasis>

Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9–10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction on enzyme activity of 70 and 20% was observed in HP and GJ extracts, respectively when protease inhibitor Pepstatin A was used. That result suggests that the main proteases in the HP were aspartic acid proteinases type (possibly Cathepsin D) and some of them were present in the GJ. Dissociating discontinuous polyacrylamide gel electrophoresis showed activity bands between 20 and 28, 30 and 34, 35 and 45, 60 and 70 kDa, and a last one between 75 and 100 kDa. We concluded that extracellular digestion of O. maya takes place in an acid environment, around pH 6. In contrast, intracellular digestion in the HP is developed at pHs between 3 and 4, where cathepsin D could be the most important enzyme for O. maya.     

The responses of digestive enzymes in juvenile and adult Rapana venosa (Valenciennes, 1846) to different temperatures

The veined rapa whelk, Rapana venosa (Valenciennes, 1846), is an important and valuable fishery species; however, it has not been routinely artificially cultured on a large scale. Here, we studied the effect of temperature (6, 16, 22 and 28°C) on the digestive enzymes in R. venosa of different sizes (Juvenile (small, S: 1.2–1.8 cm) and adult (medium, M: 4.1–5.2 cm; large, L: 7.6–8.9 cm)) by detected the enzyme activity in different digestive glands (the salivary gland, Leiblein's gland (oesophageal gland) and liver) using spectrophotometric method, to determine the optimal temperature for artificial cultivation and to explore the feeding habits of this whelk. The optimal temperature was determined to be approximately 22°C. The food intake was significantly higher at 22°C than at other temperatures, and the activity of the digestive enzyme, trypsin, was at the highest in the main digestive gland of R. venosa, the liver, at 22°C. In addition, the observed presence of cellulase and α‐amylase may suggest that carbohydrates are necessary during R. venosa development. The results of this study will help in the formulation of technical procedures for R. venosa aquaculture and in preventing its overexploitation, and explained its invasion mechanism to some extent.     

Activities of digestive enzymes in relation to ingested natural food in three carp species of the Himalayan River Ladhiya,northern India

The present investigation was carried out to study gut content and accompanying digestive enzyme profiles of the three carp species, golden mahseer Tor putitora, snow trout Schizothorax richardsonii and hill trout Barilius barila. The fishes were captured from the River Ladhiya, Champawat district, Uttarakhand, northern India. The digestive tract length was on average 1.8 and 2.3 times longer than the body, while members of Bacillariophyceae, Chlorophyceae, Cyanophyceae and Zygnematophyceae made up 95.15% and 99.63% of gut content in the golden mahseer and snow trout, respectively. In hill trout, the digestive tract being only half the body length, gut content was insects (72.73%), eggs of aquatic organisms (22.73%) and nematodes (4.55%). Estimated gut enzyme activities demonstrated that amylase activity was significantly (P < 0.05) higher in golden mahseer and snow trout compared to hill trout, while total protease, trypsin and chymotrypsin activities were all significantly (P < 0.05) higher in the hill trout compared to other species. A strong co‐relation was found among relative gut length, food composition and digestive enzyme activities of each species. This may guide the future formulation of feed to these important species of carps kept in farm.     

The effects of seasonal,sex and size on the digestive enzyme activities in freshwater crayfish (Astacus leptodactylus Eschscholtz, 1823)

The effects of seasonal, sex and size on digestive enzyme activities of Astacus leptodactylus in natural habitat were investigated in the present study. The freshwater crayfish were sampled 584 individual as seasonally from Egirdir Lake. Ten male and 10 female individuals were sampled randomly from caught crayfish in each two different size ranges (4.5–7.0, 7.1–9.9 cm) and each season. The gastrointestinal tract, gastric, intestine and midgut gland were individually dissected on ice for enzyme analysis and stomach contents. At the end of the study, amylase activities were affected by interaction of season × sex × size in intestinal, season × sex in midgut gland. However, any interaction was not determined in amylase activities of the gastric. Lipase activities were affected by interaction of season × sex × size in intestinal and gastric, season × sex in midgut gland. The protease activities in each organ were affected by interaction of season, sex and size (p ? .05). In all organs, amylase and lipase activities were similar. However, the highest protease activity was in stomach while the lowest protease activity was the intestine. The protease activities were higher than amylase and lipase activities. There is a positive correlation between protease enzyme activities in gastric with gonad maturation and active feeding period. It can be said based on data of digestive system and high protease activity that A. leptodactylus is an omnivore species in need high protein. In addition, the reason for the low levels of lipase activity may be A. leptodactylus not prefer high fat foods or not fed high fat foods.     

斑点叉尾对全植物蛋白饲料的胃排空及消化道内容物特性研究

为研究斑点叉尾(鱼回)[体质量(115.55±7.54)g]对全植物蛋白饲料的胃排空,实验比较了3种常用数学模型对其排空曲线的拟合程度,同时探究了摄食过程中消化道内容物的水分、pH和消化酶的变化规律.结果发现,3种数学模型均可较好地描述斑点叉尾(鱼回)胃排空曲线,平方根模型最适于定量描述,指数模型次之,线性模型较差.根据平方根模型分别计算得到斑点叉尾(鱼回)胃50％排空时间为12.8 h(以干重计),完全排空约36 h.在摄食后0～36 h内,内容物水分含量在后肠保持不变,在胃部呈缓慢上升趋势,而在前肠和中肠则呈先上升后下降趋势.消化道各段内容物pH在摄食后3h内均显著下降,3～30 h间总体保持平稳状态,在30 h后又呈显著上升趋势.消化道各段蛋白酶、脂肪酶和淀粉酶比活力在摄食3h内显著上升,并在3～30 h内保持相对稳定水平,摄食30 h后呈显著下降趋势.整个摄食过程中,消化道各段消化酶比活力大小依次为前肠＞中肠＞后肠＞胃.研究表明,斑点叉尾(鱼回)在摄食过程中,消化道各段内容物的水分、pH和消化酶比活力均随时间呈现相应的规律性变化.而这些内环境参数的相互作用,综合影响了特定胃排空时间的形成.     

Ontogenic changes in the digestive enzyme activities and the effect of different starvation duration on the digestive enzyme activities of larval red swamp crayfish (Procambarus clarkii)

This study was performed to determine the effect of starvation and delayed feeding on activities of digestive enzymes and alkaline phosphatase (ALP) of larval red swamp crayfish (Procambarus clarkii), so as to reveal the tolerance to prolonged starvation and the recovery of digestive enzymes after delayed feeding in larval and juvenile P. clarkii. In the control group, activities of trypsin and ALP increased significantly (p < .05) with day‐age and then kept constant at 24 days after hatching (DAH) and 10 DAH, respectively, whereas the activities of amylase and pepsin increased firstly then decreased with day‐age, and the activity of lipase increased firstly then decreased and then increased again during the development period of juvenile P. clarkii (1–31 DAH). In the group with continuous starvation (CS), activities of pepsin and lipase both decreased (p < .05) after fasting, and the activities of pepsin, lipase and trypsin in the groups with delayed feeding all increased (p < .05) and recover to the levels of the control group after food supply. However, the activity of amylase increased (p < .05) in the CS group, and it decreased to normal level after food supply. The ALP activity did not significantly (p > .05) vary after starvation, whereas it decreased in the groups with delayed feeding after 1 day of food supply, and then increased back to the level similar with the control group. Results from this study could provide information for diet preparation and feeding regime in larval and juvenile red swamp crayfish culture.     

Effects of Lactobacillus pentosus on the growth performance,digestive enzyme and disease resistance of white shrimp,Litopenaeus vannamei (Boone, 1931)

The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 10⁶, 10⁷ and 10⁸ CFU g^?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 10⁷ CFU g^?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.     

Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

From fish‐processing waste to feed additives for crayfish

This research studied the in vitro compatibility of Cherax quadricarinatus digestive proteases and crude enzyme extracts recovered from fish‐processing waste to evaluate their potential use as feed additives to increase digestion efficiency in decapod crustaceans. The sources of exogenous enzymes were: Pleoticus muelleri (Pm), Artemesia longinaris (Al), and Illex argentinus (Ia). The midgut glands of early (7.2 ± 0.34 g) and advanced juveniles (13.8 ± 1.49 g) of C. quadricarinatus were used for all the assays. The compatibility and synergy between crayfish and exogenous enzymes were evaluated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis and the hydrolysis of azocasein and four different meals. SDS–PAGE gels demonstrated that crayfish bands remained active when their enzymes were combined with the enzymes from Pm and Ia. An apparent enzymatic synergism was found when Pm and crayfish proteases were incubated together at pH 6 and 8 with azocasein (p ? 0.05). The ability to hydrolyze the different feed ingredients varied among the enzyme combinations assayed. Our findings suggest that fish waste has potential as a source of proteases, adding value to these processing residues and promoting a zero‐waste industry. Enzymes obtained from Pm and Ia are suitable to be used as food additives for crayfish. The results should be validated through feeding trials.     

Effect of probiotics on digestive enzyme activities and growth of cichlids,Etroplus suratensis (Pearl spot) and Oreochromis mossambicus (Tilapia)

In aquaculture, exposure to stressful conditions and problems related to diseases often occur resulting in serious economic loss. The use of probiotics that control pathogens through different mechanisms, aiding digestion by exoenzyme supply and establishment of beneficial microflora in the digestive tract has got increased attention recently. Two different bacterial species Micrococcus MCCB 104 and Bacillus MCCB 101 were tested as probiotics for two cichlid species Etroplus suratensis and Oreochromis mossambicus. The strains were administered separately and together, at a rate of 10³ CFU animal^?1 day^?1 for 28 days. The effects on digestive enzyme activity and growth were compared with those fed on control diet. FCR of E. suratensis was high in case of Bacillus alone as well as the mixed culture (Micrococcus and Bacillus together) than that of control. However, the SGR of E. suratensis was slightly lower than that of control during 14 days trial. SGR of both the treatments (Bacillus alone and mixed culture) was higher than that of the control on 28th day, and this might be due to the reason that specific duration is needed for the assimilation and conversion of nutrients into proteins. The intestinal digestive hydrolases of E. suratensis were profoundly enhanced mainly by Bacillus and mixed cultures. The hepatopancreatic versions of major digestive enzymes in E. suratensis were influenced variously by different probiotic treatments. The alpha‐amylase activity of hepatopancreas has been elevated significantly by mixed culture during 14 days experiments while on 28 days trials both Bacillus and mixed culture showed an enhancing effect. During 14 days feeding trials, the intestinal lipase activity has been prominently enhanced by all probiotic treatments. It is found that, in O. mossambicus, both 14 days and 28 days feeding experiments, the pepsin as well as intestinal digestive enzymes such as amylase, total alkaline protease, lipase and their hepatopancreatic counter parts showed a significant elevation in their specific activity. Intestinal alpha‐amylase and total alkaline proteases of O. mossambicus showed an increase in specific activities on both 14 days and 28 days feeding experiments with the Bacillus and Micrococcus combination. The hepatopancreatic enzymes were highly influenced by the Bacillus species in O. mossambicus. The results of this study suggest that live probiotic microorganisms such as Micrococcus MCCB 104 and Bacillus MCCB 101 may be incorporated while formulating the cost‐effective, nutritionally balanced diet of E. suratensis and O. mossambicus for better growth performance and nutrient utilization.     

Ontogenetic development of digestive tract and digestive enzymatic activities in Squaliobarbus curriculus larvae

Squaliobarbus curriculus is an economically important freshwater fish. The ontogenetic development of the digestive system of S. curriculus larvae was studied histologically and enzymatically from hatching to 30 days posthatching (DPH). Amylase, lipase, alkaline phosphatase and pepsin activities were detected from the hatching stage, indicating that these enzymes were genetically preprogrammed. Marked increases in intestinal amylase, trypsin and alkaline phosphatase activities between 10 and 20 DPH corresponded to feed acquisition and transformation. Larval development in S. curriculus could be divided into three phases: phase I (endotrophic period): 1–3 DPH; phase II (endo‐exotrophic period): 4–5 DPH; and phase III (exclusively exotrophic period): from 6 DPH onward. At hatching, the digestive tract of the larvae was an undifferentiated straight tube. On 3 DPH, the digestive tract differentiated into the mouth cavity, oesophagus and intestine. On 6 DPH, feeding was totally exotrophic and the yolk sac was completely exhausted. During the growth of S. curriculus larvae, the intestinal mucosa formed and the number of goblet cells and microvilli increased, demonstrating maturation of the digestive system. The study about the digestive development of S. curriculus larvae will contribute to better larval‐rearing strategies.     

Enzyme‐producing bacteria isolated from fish gut: a review

Digestion of food depends on three main factors: (i) the ingested food and the extent to which the food is susceptible to the effects of digestive enzymes, (ii) the activity of the digestive enzymes and (iii) the length of time the food is exposed to the action of the digestive enzymes. Each of these factors is affected by a multitude of secondary factors. The present review highlights the experimental results on the secondary factor, enzymatic activity and possible contribution of the fish gut microbiota in nutrition. It has been suggested that fish gut microbiota might have positive effects to the digestive processes of fish, and these studies have isolated and identified the enzyme‐producing microbiota. In addition to Bacillus genera, Enterobacteriaceae and Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus, unidentified anaerobes and yeast are also suggested to be possible contributors. However, in contrast to endothermic animals, it is difficult to conclude the exact contribution of the gastrointestinal microbiota because of the complexity and variable ecology of the digestive tract of different fish species, the presence of stomach and pyloric caeca and the relative intestinal length. The present review will critically evaluate the results to establish whether or not intestinal microbiota do contribute to fish nutrition.     

Effect of incubation temperature on carbohydrate digestion in important teleosts for aquaculture

The activity and capacity (activity × tissue weight) of digestive carbohydrases (total carbohydrase, α‐glucosidase, α‐amylase) was examined in vitro under a range of (more realistic) incubation temperatures (5, 18 and 25°C) and pH (7.6) more likely to be encountered during rearing, along the digestive tract of important teleost species for aquaculture (Dicentrarchus labrax, Sparus aurata, Oreochromis aureus, Oncorhynchus mykiss and Salmo salar). Results indicate, among other things, an overestimation of digestive carbohydrase levels when performing assays at 37°C, a different effect of temperature on digestive enzyme performance along the digestive tract of examined species, and the increased importance of α‐glucosidase towards carbohydrate digestion. Implications regarding the capacity of each species for carbohydrate digestion in nature, as well as feed manufacturing, are discussed.     

九孔鲍性腺发育过程中消化酶活力的变化

用酶学方法研究了性腺发育过程中九孔鲍肝胰腺和消化道中蛋白酶、淀粉酶和纤维素酶活力的变化。试验结果表明,雌、雄九孔鲍各消化酶活力均随着性腺发育呈先升后降的变化趋势。在性腺发育早期和晚期酶活力较低,在性腺发育中期酶活力显著升高,且均在成熟前期达到最高值。九孔鲍繁殖期各消化酶的活力分布存在器官特异性和性别差异,九孔鲍的淀粉酶和纤维素酶活力在各性腺发育期均表现为肝胰腺消化道,雌鲍雄鲍。同种消化器官内各消化酶的活力也存在差异,雌、雄九孔鲍繁殖期肝胰腺和消化道内各消化酶的最大活力均为:纤维素酶淀粉酶蛋白酶。     

Analysis of bacterial community and bacterial nutritional enzyme activity associated with the digestive tract of wild Chilean octopus (Octopus mimus Gould, 1852)

Information on the bacterial community associated with octopus is very scarce, unlike fish and other molluscs. This study revealed the bacterial community associated with digestive tract of wild Chilean octopus Octopus mimus using a culture‐dependent method and 16S rDNA clone library. Moreover, we analysed the bacterial nutritional enzyme activity of culturable bacteria. A culture‐dependent method showed that the composition of the culturable bacterial community was substantially different between female and male octopus. The predominant species in female octopus were Vibrionaceae and Streptococcaceae, whereas only Vibrionaceae was dominated in male octopus. Bacterial nutritional enzyme activities of culturable bacteria from male octopus were much higher than female octopus. The 16S rDNA clone library analysis showed that the bacterial community of male octopus exhibited a higher diversity than that of female octopus. The genus Mycoplasma was the predominant bacteria in the digestive tract of all octopus samples. The results obtained in this study raise the possibility that each octopus has different food consumption due to different bacterial community and nutritional enzyme activity, although Mycoplasma sp. is one of the predominant bacteria in the digestive tract. Moreover, our results are useful for the future of microbiological investigation associated with the octopus and for probiotics in the octopus aquaculture.     

Exogenous proteinases as feed supplement for shrimp: in vitro evaluation

To test the efficacy of adding enzyme supplements to feeds, an in vitro study was conducted by mixing bovine trypsin or proteinases from the gastric juice of the Cortez swimming crab Callinectes bellicosus with an enzyme extract from the digestive gland of the whiteleg shrimp Penaeus vannamei. Enzymes alone and mixtures were tested for hydrolysing proteinaceous natural substrates (bovine casein, bovine haemoglobin, and bovine serum albumin). All enzyme preparations hydrolysed casein. Shrimp enzymes hydrolysed haemoglobin but not serum albumin. Bovine trypsin and crab proteinases hydrolysed serum albumin but not haemoglobin. The mixture of shrimp and crab enzymes generated more hydrolytic products of serum albumin than shrimp enzymes alone. Shrimp enzymes mixed with bovine trypsin did not hydrolyse albumin because the bovine trypsin vanished; shrimp enzymes hydrolysed bovine trypsin. Results indicated that it is naive to assume that proteinolytic enzymes from different species will add their catalytic capabilities if mixed; here, we demonstrated that they may act as proteinases and will hydrolyse available protein regardless of its function. Our conclusion is that enzyme supplements should be tested in vitro before intending them as exogenous proteinases in feeds. This technique can be used to assess the compatibility and additivity of proteinases when mixed for biotechnological purposes. Besides, the technique can demonstrate who hydrolyses whom.     

Effects of supplementation of decapod zoea to Artemia basal diet on fatty acid composition and digestive gland histology in common octopus (Octopus vulgaris) paralarvae

The present study aimed to evaluate the effect of the supplementation of different crab zoeas to enriched Artemia basal diet for Octopus vulgaris paralarvae during the first month of life. Paralarvae were fed using enriched Artemia nauplii alone and Artemia co‐fed either first zoea stages of Grapsus adscensionis or Plagusia depressa. The experiment was carried out over a period of 28 days, in 0.12 m³ tanks with a flow‐through rearing system. Growth in dry weight as well as mantle length and width were assessed weekly. Additionally, prey and paralarvae fatty acid composition and digestive gland (DG) histology were evaluated. Addition of low amounts of crab zoeas (approx. 100 indv. L^?1 day^?1) provided during critical life stages of O. vulgaris proved to be good enough to improve paralarvae growth and survival in comparison with those fed exclusively on enriched Artemia. These results were supported by the finding of a higher number of glycoprotein absorption vacuoles in the DG from paralarvae co‐fed crab zoeas, suggesting a higher feeding activity. In addition, fatty acid analysis of crab zoea showed that these are good sources of dietary arachidonic and eicosapentaenoic acids during the octopus planktonic life stage, whereas the low docosahexaenoic (DHA) content suggests the use of additional DHA sources or higher zoea densities to meet paralarvae nutritional demand to carry out a successful metamorphosis to benthic life.     

Ontogenetic changes in digestive proteases and carbohydrases from the Australian freshwater crayfish, redclaw Cherax quadricarinatus (Crustacea, Decapoda, Parastacidae)

Juvenile redclaw crayfish, Cherax quadricarinatus, show non‐selective feeding behaviour when commencing feeding, but change to a dietary preference for plant material as they grow. This study aimed to find any ontogenic changes in digestive enzymes that might be associated with the change of diet. The enzymes in the digestive tract initially showed high levels of proteases and low levels of carbohydrases, but as the crayfish grew, the levels of proteases fell and the levels of the carbohydrases generally rose, in parallel with the increased preference for plant food. Cellulase was present in all free‐living stages. Dietary requirements of juvenile redclaw are probably quite similar to those of larger specimens.