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74 The Open Fish Science Journal, 2009, 2, 74-86

Open Access

Digestive Enzymes in Some Teleost Species of Interest for Mediterranean


Aquaculture

G. Caruso, M.G. Denaro* and L. Genovese

National Research Council, Istituto per l’Ambiente Marino Costiero, Messina, Italy
*University of Messina, Department of Life Sciences “M. Malpighi”, Faculty of Sciences, Messina, Italy

Abstract: In intensive aquaculture, basic studies on fish digestive capacity and metabolism play a key role for diet formu-
lation. In particular, intensive rearing of a new species is strictly related to the development of appropriate nutritional pro-
tocols and therefore knowledge of specific features concerning physiology and nutrition is needed. Nutrient utilization by
fish is a direct function of the availability of suitable digestive enzymes along the gastro-intestinal tract. Therefore the de-
termination of specific enzymatic activities (proteases, carbohydrases and lipases) may provide qualitative information
about the digestive capacity and the efficiency of each reared species to use feeding components. Recent observations on
digestive enzymatic profiles and on digestive ability of some Teleost species with large aquaculture potential and recog-
nized as new candidates for product diversification have been reviewed in this paper. Attention has been given to the dif-
ferent features (dietary composition, digestive phases, life stages, contribution of bacterial .enzymes) that may affect en-
zymatic patterns.

Keywords: Digestion, enzymes, fish, diversification, new candidate species.

INTRODUCTION feeding habit that is reflected in different anatomical and


functional features; both nutritional and physiological char-
Expansion of fish farming is strictly related to improve-
acteristics allow them the exploitation of a wide range of
ment in nutrition, fish welfare, and husbandry practices.
food resources, thus improving their adaptation to changing
Each of these topics represents a challenge for future devel-
environmental conditions.
opment in aquaculture. Mass production of new species de-
pends strongly on the elaboration of proper feeding protocols Several studies [3-8] have previously shown that the dis-
able to satisfy their nutritional requirements. Studies aimed tribution and activity of digestive enzymes within the gut are
at investigating the functioning of the digestive tract in dif- affected by feeding habits. Herbivorous fish usually possess
ferent species can provide relevant tools for the optimisation a gut longer than carnivores and a digestive enzymatic pro-
of the relative percentage of their dietary macronutrients; file adapted to feed ingredients, with the predominance of
therefore, knowledge of digestive enzymes of fish has im- carbohydrases (i.e. amylases, disaccharidases), which allow
portant practical implications for their nutrition. them the hydrolysis of polysaccharides. Conversely, carnivo-
rous fish have a short intestine, with higher levels of prote-
The ability of fish to metabolise a diet depends on the
ases compared with herbivorous fish, while also amylase
availability of appropriate digestive enzymes, which mediate
[6, 9] and lipase [10] are represented in minor percentage in
specific degradation pathways, as well as on both physical
their digestive tract. In fact, in the dietary composition of the
and chemical nature of food [1]. The measurement of spe- most carnivorous fish, proteins and lipids play an important
cific activities (proteases, carbohydrases and lipases) may
role as a source of basic nutrients and energy [11]. Moreo-
provide information about the whole digestive capacity and
ver, a differentiation in enzymatic distribution within the
the efficiency of species reared to use feeding components
digestive organs has also been observed; while food ingested
[2].
is first hydrolysed in the stomach by the acid protease pep-
Digestion is a progressive process, that starts in the stom- sin, alkaline enzymes (i.e. trypsin, chymotrypsin, car-
ach and continues to take place along the intestine. By this boxypeptidases, lipases) from the pancreas and the liver al-
process, ingested materials are hydrolysed into smaller size low the progression of food digestion inside the midgut
molecules, such as amino acids, simple sugars and fatty ac- [3, 12, 13].
ids produced by the hydrolysis of proteins, sugars or lipids,
Until now, comparative studies of the digestive enzymes
respectively. Fish usually display high versatility in their
in different fish species have been performed [6, 14-15];
nevertheless, fish digestive capabilities have not yet been
completely investigated and this explains some difficulties in
*Address correspondence to this author at the National Research Council, formulating dietary protocols responsive to fish metabolism.
Istituto per l’Ambiente Marino Costiero, Spianata S. Raineri, 86 – 98122 In particular, the inter-relationships between enzyme expres-
Messina, Italy; Tel: +39-090-669003; Fax: +39-090-669007;
E-mail: [email protected] sion and control factors, such as the adaptive responses of

1874-401X/09 2009 Bentham Open


Digestive Enzymes in New Teleost Species for Aquaculture The Open Fish Science Journal, 2009, Volume 2 75

the digestive enzymatic spectrum in relation to the dietary Pagellus bogaraveo, belonging to Sparidae, is a species
components or life stage, are still not clearly understood, widely distributed in different temperate environments, in-
stressing the importance of studies addressing these topics. cluding from inshore waters, above various types of seabed
(rocks, sand, mud) to 400 m (Mediterranean Sea) and 700 m
Several studies, performed in recent years in our labora-
(Atlantic Sea). Young specimens are found near the coast,
tory, have focused on physiological and nutritional aspects
while adults inhabit the continental slope especially over
related to fish farming. Particular attention has been given to
muddy seabeds. It is omnivorous, but it prefers crustaceans,
the assessment of digestive abilities in fish species consid-
molluscs, worms and small fish; it displays rearing charac-
ered as “emerging” in aquaculture, in order to verify their
teristics (i.e. fast growth) similar to other Pagellus species
potential as new candidates for aquaculture diversification. [33] and represents an important human food source.
In fact, most of these species have high commercial value
and/or offer interesting perspectives for market expansion Blackspot seabream (Pagellus erythrinus) is a popular
especially in the Mediterranean area; nevertheless, only a table fish recently regarded as a new candidate species for
few data on nutrient digestion are available in literature aquaculture. It is found on inshore waters, on various sea-
[6, 16-19]. beds (rock, gravel, sand and mud) to 200 m (Mediterranean
Sea) or 300 m (Atlantic Sea), moving to deeper waters dur-
The aim of the present review is to improve current ing winter. It is omnivorous, but feeds mainly on benthic
knowledge of the amount and distribution of digestive en- invertebrates and small fish. Despite a growing commercial
zymes mostly in species belonging to Sparidae family (axil- interest, only few studies regarding its biology, reproduction
lary seabream, blackspot seabream, common pandora), but and larval rearing have been carried out [34, 35]; in particu-
also species of Carangidae (greater amberjack) and Serrani- lar, the structural and biochemical modifications occurring
dae [dusky grouper Epinephalus marginatus (Lowe) and during the ontogenesis of the digestive tract have never been
grouper, Epinephalus aeneus (Geoffrey St Hilaire)] were investigated.
included in the research. In particular, some factors that may
affect enzymatic patterns were investigated, such as: Dusky grouper (Epinephalus marginatus) and grouper
(E. aeneus) are both carnivorous predators, feeding mainly
-nature and composition of the diet, such as observation on fish and molluscs. Concerning their ecological habitat,
made in axillary seabream, Pagellus acarne (Risso) [20, 21] adult specimens are usually found on rocky or muddy-sandy
and in blackspot seabream, Pagellus bogaraveo (Brünnich) seabeds, at depths between 20-200 m, whereas juveniles are
[22, 23]. frequently found in coastal lagoons and estuaries.
-digestive phases, such as those studied in greater amber- Experimental Design and Samples Collection
jack Seriola dumerilii (Risso) [24, 25] as well as in blackspot
seabream [22]. The study was carried out at the facilities of the Institute
for Coastal Marine Environment of Messina.
-possible interaction between native, endogenous, en-
zymes and exogenous enzymes produced by microbial flora, For the experiments, young specimens of greater amber-
such as that investigated in greater amberjack [26] and in jack were caught with a circular net in summer, then accli-
blackspot seabream [27]. mated (1-2 days) in floating cages before their transport to
the Institute. They were kept in a 12 m3 PVC tank and fed an
-life stages, with reference to changes occurring in enzy- experimental live diet (Trachurus trachurus) supplemented
matic patterns during growth (as observed in larvae of com- with a commercial mineral-vitamin mixture, which was ad-
mon pandora, Pagellus erythrinus L [28]. ministered until satiation [25]. The experiment on this spe-
-specific diversity, concerning some species belonging to cies was carried out on adult specimens (average weight: 500
Sparidae [20-22, 29] and Serranidae families. ± 72.15 g) in October 1994 (temperature, 18°C), when fish
were in an active phase of alimentation. Five fish were sacri-
MATERIALS AND METHODOLOGY ficed at different time intervals from food ingestion, namely
2, 4 and 6 hours after, as well as after 2 days of starvation
Notes on the Biology of the Examined Species (fish taken as a control).
Greater amberjack (Seriola dumerilii) is a Carangidae Specimens of axillary seabream were caught from the
species living in the Mediterranean Sea. It inhabits deep Straits of Messina in summer 1993 and acclimated for 15
seaward reefs, occasionally entering coastal bays; it is a car- days in cylindro-conical tanks having a capacity of 1.6 m3,
nivorous species, feeding primarily on fishes and inverte- with two daily changes of water and no forced aeration. Ju-
brates. A nursery area for this species is the Aeolian Archi- veniles (mean initial body weight 45.6 ± 2.6 g) were divided
pelago in Sicily. in two experimental groups at a density of 8 fish m-2 and fed
Axillary seabream (Pagellus acarne), belonging to throughout the experiment with two diets: diet A, based on
Sparidae, is a carnivorous fish widely distributed in the fish offal consisting of Trachurus trachurus supplemented
Mediterranean Sea; in the Straits of Messina it is frequent in with vitamins and mineral salts, and diet B, consisting of
sandy or muddy seabeds [30]. Juvenile stages occur in commercial dry pellets for seawater carnivorous fish
coastal environments, whereas adult specimens have pelagic (TROUW NUTRITION SpA, Verona, Italy), administered
and gregarious habits [31]. The high commercial value and daily at 20 and 10% of the fish live weight.
the enhanced rhythm of growth make this fish a suitable spe- The composition of the basal diets (as percentage on dry
cies for intensive rearing [32]. basis) was: diet A, crude protein: 74.01, lipid 15.50, ash,
76 The Open Fish Science Journal, 2009, Volume 2 Caruso et al.

11.22; diet B, crude protein: 35.87, lipid 12.53, crude fibre intestine), having cure of avoiding reciprocal contamination.
2.20, N-free extract, 20.60). After being weighted, they were homogenized with Potter in
Tris buffer 50 mM pH 7.0 (a volume 5 times the sample
Specimens were collected from each group, during Octo-
weight, w/v), and then centrifuged at 3000 rpm for 10 min-
ber, when water temperatures ranged from 18.4 to 19.4 °C.
utes. The supernatant obtained was used as an enzymatic
Concerning blackspot seabream, a first experiment was extract for further biochemical determination. All the opera-
performed in 2003 on reared juveniles (average weight: 91.6 tions were carried out at a temperature lower than +5°C.
± 12.5 g), in the framework of a research study aimed at
For enzymatic measurements in larvae, specimens were
evaluating the effects of different vegetable oils on the
sampled immediately before the food distribution, pooled
growth performances of this fish species. Juveniles were
and immediately stored at -80°C until assays. A sample of
divided in three different groups and fed with different ex-
food was also taken as a control. All the samples were ho-
perimental diets, indicated as “fish”, “linum” and “echium”.
mogenised in 5 volumes (v/w) of ice-cold distilled water,
“Fish” diet consisted of a standard diet containing fish oil
according to the protocol of Ribeiro et al. [37]; whole larvae
(fish flour 55%, corn flour 20%, oil 7.5%, crude starch 6.5%,
were used for younger specimens, while from day 31, the
extruded soybean flour 5.5%, crude fibre 5.5%, mineral salts
heads were removed and the remaining body portions
3%); “linum” and “echium” diets had been obtained by sub-
pooled.
stituting the fish oil contained in the “fish” diet with linum
and echium oils. A fourth group was fed a commercial dry Analytical Procedures for the Determination of Digestive
diet for carnivorous fish (HENDRIX S.p.A., Mozzecane, Enzymes
Verona, Italy), and taken as a control. Diets were adminis-
tered as 1.2% of the total body biomass calculated weekly. For the enzymatic assays on adult and juvenile speci-
From each group, five specimens were sacrificed after 2 days mens, a group of 5 fish were used for each species, while a
of starvation as well as 4 hours after feeding, with the aim of total of 100 larvae were sacrificed to study the enzymatic
studying changes in enzymatic distribution due to the pro- distribution. Standard procedures for the determination of
gression of digestion. pepsin, trypsin and chymotrypsin, carboxypeptidase A and
B, leucine aminopeptidase, elastase, amylase, lipase, were
A second experiment was carried out on blackspot se- followed. In particular, the substrates used to measure the
abream juveniles (average weight: 66.85 ± 17.65 g) in 2005, activity of each proteolytic enzyme were the following: bo-
in order to investigate the effects induced on the digestive vine haemoglobin (SIGMA-ALDRICH) for the determina-
enzymes by the administration of two diets containing dif- tion of pepsin activity [38]; N-toluen-sulphonyl-L-arginine
ferent levels (20% and 35%) of rice protein concentrate as a methyl ester and N-benzoyl-L-tyrosine ethyl ester (SIGMA-
protein source, in partial substitution of fish meal [23]. ALDRICH) as the substrates for trypsin and chymotrypsin
In common pandora, digestive enzymes were investi- [39], respectively; L-hippuryl-L-phenylalanine and L-
gated at larval as well as at juvenile stages. Larvae were pro- hippuryl-L-arginine (SIGMA-ALDRICH) for carboxypepti-
vided by COISPA (Bari, Italy), where they were intensively dases A and B [40]. Elastase and leucine aminopeptidase
reared (initial density of  150 larvae x l –1 ; tank volume of were detected in the gastro-intestinal tracts of greater amber-
1.5 m3, temperature range: 18-19°C and a natural photope- jack, axillary seabream, common pandora and grouper only,
riod) using pseudogreen water based on a mixture of micro- measuring the hydrolysis of L-leucyl-naphthyl-amide and
algae Isochrysis galbana and Tetraselmis suecica [36]. Since elastin-orcein (SIGMA-ALDRICH), respectively [41, 42].
the 3rd Day After Hatching (DAH), diet was supplemented Total proteases were measured using a non-specific assay
with Rotifers (Brachionus plicatilis; small strain), enriched (Kunitz’ s method [43], and its further modification [44]),
in their fatty acid content through commercial emulsifiers based on the use of casein (SIGMA-ALDRICH, St. Louis,
USA) as the substrate. Amylase was determined using solu-
(DHA SELCO, InVE AQUACULTURE, Dendermonde,
ble starch (SIGMA-ALDRICH) as the substrate [45]. Lipase
Belgium). Further changes in the dietary composition were
activity was measured using a kit (SIGMA-ALDRICH)
gradually performed, with some overlapping phases. Artemia
based on a titrimetric method [46]. For all the enzymatic
nauplii were administered at the 28th day while the artificial
activities, values obtained were normalised to the protein
food (Ø 544 μm) was introduced from the 35th day onwards.
content of each sample, as estimated by the Lowry’s method
Enzymatic measurements were performed at 0, 3, 6, 10, 17,
[47], and expressed as specific activities (Units per mg of
24, 31, 38 and 45 DAH (days after hatching). protein, U mg-1 protein). Data were analysed statistically by
A further study was carried out in 1999 on the digestive ANOVA test. Differences were considered statistically sig-
tract of common pandora juveniles (average weight: 200 ± nificant when p  0.05.
19 g) feed with an extruded diet (HENDRIX) [29].
Enzymes from Bacterial Strains
In dusky grouper and grouper, digestive enzymes were
studied on juveniles of 150 ± 35 g (average weight) sampled Bacterial strains isolated from the gastro-intestinal tracts
after 2 days of starvation. were tested for their biochemical capabilities to hydrolyse
different organic compounds by culture method on plates of
Treatment of the Samples specific media. In particular, protease activity was deter-
mined using casein as the substrate (Skim milk at 2% final
For the preparation of crude enzymatic extracts, whole concentration) and measuring the diameter of the clearing
digestive tracts were removed from fish specimens and di- zones produced after incubation at 35°C; gelatinase was as-
vided into different portions (stomach, pyloric caeca and
Digestive Enzymes in New Teleost Species for Aquaculture The Open Fish Science Journal, 2009, Volume 2 77

sayed by streaking cultures onto Bacto Nutrient gelatin Table 1A. Enzyme Values (mean ± s.d., n=5) Measured at Star-
(Oxoid) plates. Lipase production was tested using Tween 80 vation in Blackspot Seabream Pagellus bogaraveo fed
(polyoxyethylene sorbitan mono-oleate) as a substrate, at a Diet Containing Linum, Fish, Echium Oils, Com-
1% final concentration. The ability to hydrolyse starch was pared to a Control Diet. Values are Expressed as U
assayed in a culture medium containing 1% starch as the mg-1 Protein
substrate. Activity against elastin was determined after incu-
bation of a liquid culture of bacterial cells with elastin congo At Starvation
Diet Enzyme
red and further spectrophotometric measurement of the opti- Stomach Pyloric Caeca Intestine
cal absorbance of the supernatant fluid at 488 nm. Chitinase
was determined using plates of a medium containing 2% Linum Total proteases 0.361 ± 0.01 0.582 ± 0.005 0.221 ± 0.001

shrimp and crab shell powder as the major carbon source. Pepsin 0.188 ± 0.002 0.00 ± 0.00 0.00 ± 0.00

Trypsin 0.00 ± 0.00 0.084 ± 0.029 0.074 ± 0.043


RESULTS
Chymotrypsin 0.00 ± 0.00 0.082 ± 0.015 0.001 ± 0.0003
Influence of Dietary Composition and Nutritional Habit
Carboxypeptidase A 0.0002 ± 0.0001 0.0002 ± 0.0004 0.037 ± 0.009
The nature and composition of the diet administered
Carboxypeptidase B 0.00 ± 0.00 0.005 ± 0.001 0.002 ± 0.003
strongly affects the metabolic capabilities of fish, so that the
administration of different diets may yield to a diversifica- Amylase 0.34 ± 0.02 0.53 ± 0.03 0.383 ± 0.040

tion in the distribution of digestive enzymes. This issue, in- Lipase 0.00 ± 0.00 0.40 ± 0.10 2.0 ± 0.10
vestigated in axillary seabream specimens divided into ex-
Fish Total proteases 1.33 ± 0.01 8.567 ± 0.002 3.55 ± 0.002
perimental groups and fed a protein-rich diet compared with
a non-proteic one, showed that enzymatic profiles reflected Pepsin 0.119 ± 0.008 0.00 ± 0.00 0.00 ± 0.00

dietary composition. In fact, a diet rich in proteins, such as Trypsin 0.00 ± 0.00 0.286 ± 0.030 0.051 ± 0.038
fish offals (diet A), resulted in higher amounts of proteases
Chymotrypsin 0.00 ± 0.00 0.145 ± 0.081 0.381 ± 0.010
in the specimens assayed, while amylase and lipase levels
were greater in fish fed diet B, containing higher percentages Carboxypeptidase A 0.009 ± 0.01 0.080 ± 0.060 0.012 ± 0.002
of non-proteic compounds in its composition (Fig. 1). Carboxypeptidase B 0.0005 ± 0.00 0.005 ± 0.002 0.018 ± 0.012

In nutritional research, another interesting issue concerns Amylase 0.36 ± 0.02 0.463 ± 0.04 0.345 ± 0.03
the replacement of fish oil with vegetable oil, regarded as Lipase 0.00 ± 0.00 0.40 ± 0.10 0.40 ± 0.10
important dietary ingredient. This consideration lead us to
investigate the effect on the digestive enzymes of blackspot Echium Total proteases 3.03 ± 0.07 20.54 ± 0.005 13.07 ± 0.02

seabream of linum and echium seed oils, included in the die- Pepsin 0.475 ± 0.017 0.00 ± 0.00 0.00 ± 0.00
tary composition, as a source of polyunsaturated fatty acids Trypsin 0.00 ± 0.00 0.132 ± 0.010 0.082 ± 0.040
(n-3). No significant differences in the behaviour of diges-
tive enzymes were observed at starvation as well as 4 hrs Chymotrypsin 0.00 ± 0.00 0.064 ± 0.014 0.086 ± 0.012

after feeding (Tables 1A, B). Carboxypeptidase A 0.00 ± 0.00 0.058 ± 0.010 0.005 ± 0.010

Carboxypeptidase B 0.019 ± 0.013 0.009 ± 0.0011 0.008 ± 0.003


60
stomach A stomach B Amylase 0.08 ± 0.005 0.121 ± 0.010 0.135 ± 0.01
pyloric caeca A pyloric caeca B Lipase 0.00 ± 0.00 0.40 ± 0.10 0.80 ± 0.30
intestine A intestine B
Control Total proteases 1.289 ± 0.001 7.85 ± 0.006 6.97 ± 0.005
40
U mg-1protein

Pepsin 0.363 ± 0.025 0.00 ± 0.00 0.00 ± 0.00

Trypsin 0.00 ± 0.00 0.028 ± 0.005 0.071 ± 0.005

Chymotrypsin 0.00 ± 0.00 0.546 ± 0.022 0.072 ± 0.012


20
Carboxypeptidase A 0.00 ± 0.00 0.103 ± 0.040 0.085 ± 0.060

Carboxypeptidase B 0.018 ± 0.017 0.017 ± 0.007 0.026 ± 0.002

Amylase 0.09 ± 0.02 0.139 ± 0.03 0.104 ± 0.030


0
T
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A
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L
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Lipase 0.00 ± 0.00 2.0 ± 0.5 0.40 ± 0.10


A
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feeding); this depressive effect could induce alterations in


Fig. (1). Average values of digestive enzymes detected in the or-
the preliminary, gastric, breakdown of dietary components,
gans of specimens (n= 5) of axillary seabream Pagellus acarne resulting in the elongation of digestive times. In the same
(Risso) fed with diet A (fresh offals) compared with those measured group (fish fed with fish oil), trypsin content in the intestine
in specimens fed with diet B (non-proteic diet). decreased, although not at a significant level, compared with
the control group. Diet containing linum oil caused a de-
Fish fed a diet supplemented with fish oil showed a sig- crease in chymotrypsin values in the pyloric caeca and in the
nificant reduction of pepsin content in the stomach, com- intestine; in this organ, an increase in lipase content oc-
pared with fish fed echium oil (F=7.02, P < 0.05, 4hrs after curred, while the carboxypeptidase B levels were signifi-
78 The Open Fish Science Journal, 2009, Volume 2 Caruso et al.

Table 1B. Enzyme Values (Mean ± s.d., n=5) Measured 4 Hours The effects produced on the digestive enzymes by the in-
after Feeding in Blackspot Seabream Pagellus boga- clusion of the rice protein concentrate in the diet of blackspot
raveo Fed Diet Containing Linum, Fish, Echium oils, seabream are reported in Fig. (2).
Compared to a Control Diet. Values are Expressed
as U mg-1 Protein
8
stomach A stomach B stomach C
After Feeding (4hrs) 7
Diet Enzyme pyloric caeca A pyloric caeca B pyloric caeca C
Stomach Pyloric Caeca Intestine 6
intestine A intestine B intestine C

U mg protein
Linum Total proteases 0.517 ± 0.03 0.896 ± 0.002 0.489 ± 0.001 5

-1
4
Pepsin 0.688 ± 0.014 0.00 ± 0.00 0.00 ± 0.00
3
Trypsin 0.00 ± 0.00 0.088 ± 0.017 0.045 ± 0.007
2
Chymotrypsin 0.00 ± 0.00 0.090 ± 0.055 0.007 ± 0.040
1
Carboxypeptidase A 0.004 ± 0.02 0.013 ± 0.07 0.053 ± 0.040
0
Carboxypeptidase B 0.0002 ± 0.0001 0.041 ± 0.010 0.011 ± 0.009

in

e
in

se
si

as
s
ps

e
yp

pa
as

as

yl
ry
Pe

Tr

Li
Am
ot

tid

tid
Amylase 0.40±0.05 0.445 ± 0.040 0.568 ± 0.05

ep

ep
hy

yp

yp
C

ox

ox
b

b
Lipase 0.00 ± 0.00 3.20 ± 0.30 2.0 ± 0.2

ar

ar
C

C
Fish Total proteases 1.944 ± 0.001 13.72 ± 0.004 12.03 ± 0.005

Pepsin 0.059 ± 0.021 0.00 ± 0.00 0.00 ± 0.00


Fig. (2). Digestive enzymes recorded in blackspot seabream Pagel-
Trypsin 0.00 ± 0.00 0.033 ± 0.007 0.013 ± 0.017 lus bogaraveo (Brünnich) specimens (n=5) fed diets A (control
diet), B and C, containing 0, 20 and 35% rice protein concentrate,
Chymotrypsin 0.00 ± 0.00 0.140 ± 0.042 0.108 ± 0.042
respectively.
Carboxypeptidase A 0.00 ± 0.00 0.099 ± 0.050 0.064 ± 0.009

Carboxypeptidase B 0.184 ± 0.104 0.153 ± 0.027 0.082 ± 0.065 Fish fed with the diet containing the highest level of rice
protein concentrate (35%) displayed a significant increase of
Amylase 0.43 ± 0.04 0.441 ± 0.037 0.452 ± 0.030
pepsin in the stomach (F = 448.64, P < 0.01) and of trypsin
Lipase 0.00 ± 0.00 5.60 ± 0.60 0.80 ± 0.10 in the intestine (F = 18.57, P< 0.01) (Fig. 2). The same
Echium Total proteases 7.84 ± 0.06 24.36 ± 0.09 17.51 ± 0.003 stimulating effect was also observed with the diet containing
20% of rice protein concentrate. The contents of chymotryp-
Pepsin 0.847 ± 0.005 0.00 ± 0.00 0.00 ± 0.00
sin, carboxypeptidase A and B values were significantly en-
Trypsin 0.00 ± 0.00 0.073 ± 0.002 0.110 ± 0.013 hanced in the intestine of fish fed the diet containing 35% of
Chymotrypsin 0.00 ± 0.00 0.431 ± 0.059 0.144 ± 0.024 this compound (F versus control diet = 29.40, 121.50, 15.48,
P < 0.01, respectively). Therefore, data obtained suggested
Carboxypeptidase A 0.00 ± 0.00 0.053 ± 0.020 0.041 ± 0.03
the inclusion of this compound as a dietary ingredient for its
Carboxypeptidase B 0.00 ± 0.00 0.087 ± 0.072 0.089 ± 0.018 positive effects on protein digestibility in blackspot se-
Amylase 0.12 ± 0.01 0.234 ± 0.02 0.276 ± 0.022 abream.
Lipase 0.00 ± 0.00 6.40 ± 0.50 2.80 ± 0.20
Effect of the Chronobiology of the Digestion
Control Total proteases 3.04 ± 0.05 15.51 ± 0.03 11.58 ± 0.03
In adult specimens of greater amberjack, acid protease of
Pepsin 0.293 ± 0.004 0.00 ± 0.00 0.00 ± 0.00
the stomach (pepsin) displayed values of 123 and 124.50 U
Trypsin 0.00 ± 0.00 0.141 ± 0.003 0.079 ± 0.002 mg-1protein at starvation and 4 hrs after feeding (Fig. 3A). At
Chymotrypsin 0.00 ± 0.00 0.416 ± 0.096 0.07 ± 0.04
the same digestive phases, alkaline proteases were 142.50
and 152.50 Umg-1protein in the pyloric caeca and 137.50 and
Carboxypeptidase A 0.00 ± 0.00 0.118 ± 0.12 0.119 ± 0.010 150 U mg-1protein in the intestine, respectively. In all the
Carboxypeptidase B 0.017 ± 0.013 0.108 ± 0.110 0.113 ± 0.017 examined organs, the levels of proteases 6 hrs after feeding
were similar to those measured in an empty stomach (Fig.
Amylase 0.13 ± 0.01 0.273 ± 0.030 0.195 ± 0.020
3A). Leucine aminopeptidase content varied from 53.5 to
Lipase 0.00 ± 0.00 3.60 ± 0.30 1.60 ± 0.20 84.5 U mg-1protein in the stomach, respectively at starvation
and 4 hrs after feeding; from 121.0 to 129.0 U mg-1protein in
cantly reduced (F=31.69, P < 0.01, at starvation). Compared the pyloric caeca and from 99.0 to 116.5 U mg-1 protein in
with the control, both linum and fish oils increased signifi- the intestine, respectively (Fig. 3A).
cantly amylase levels, particularly in the stomach at starva- Elastase content varied from 0.03 to 1.00 U mg-1protein
tion (F=40.80 and 57.63, P < 0.01 respectively). This result in the stomach; from 1.50 to 2.30 U mg-1protein in the pylo-
suggested that the vegetable nature of the linum oil was ef-
ric caeca and from 0.65 to 1.55 U mg-1protein in the intestine
fective in stimulating the digestive capabilities of the carbo-
(Fig. 3B).
hydrate component.
Digestive Enzymes in New Teleost Species for Aquaculture The Open Fish Science Journal, 2009, Volume 2 79

At the same sampling conditions, lipase activity was 3.30


200 and 3.75 U mg-1 protein in the pyloric caeca and 5.35 and
4.00 U mg-1 protein in the intestine (Fig. 3D).
150

100

U mg-1protein
10
50
5
0
0
0 2 time (hrs) 4 6 0 2 4 6
Total pro teases-sto mach time (hrs)
Total pro teases - pyloric caeca pyloric caeca intestine
Total pro teases - intestine Fig. (3D). Average values of lipase measured in the organs of
greater amberjack Seriola dumerilii (Risso) specimens fed with
Leucine Aminopeptidase -stomach fresh offals. See Fig. 3A for caption.
Leucine Aminopeptidase - pyloric caeca
Leucine Aminopeptidase - intestine Contribution of Bacteria to Fish Digestive Capability
Estimations, carried out in the stomach of greater amber-
Fig. (3A). Average values of total proteases and leucine aminopep- jack, of the specific composition of bacterial flora and of its
tidase measured at time 0, after 2 days of starvation, and 2, 4, 6 hrs capacity to degrade organic macromolecules (Figs. 4a, A)
after feeding, in the organs of greater amberjack Seriola dumerilii showed that, in this fish species, on average 30% of gastric
(Risso) specimens (n=5) fed with fresh offals. bacteria play a significant role in the digestive capability. A
similar result was observed in the intestine (Figs. 4b, B),
3,0 where bacterial decomposition was particularly enhanced
U mg-1 protein

with respect to the proteolytic activity towards gelatin, ca-


2,0 sein and elastin.
The high density of bacteria able to degrade complex
1,0 molecules (average value 105 CFU g-1 of homogenate)
pointed out how microorganisms were actively involved in
0,0 the breakdown of organic substrates into simple molecules
within the digestive tract of fish.
0 2 4 6
a
time (hrs)
100
stomach pyloric caeca intestine
80

Fig. (3B). Average values of elastase measured in the organs of 60


%

greater amberjack Seriola dumerilii (Risso) specimens fed with 40


fresh offals. See Fig. 3A for caption. 20
0
Amylase showed values of 0.04 and 0.35 U mg-1 protein 1h 2h 4h
in the stomach, 0.28 and 0.40 U mg-1 protein in the pyloric Pseudomonas Micrococcus Aeromonas
caeca and 0.18 and 0.35 U mg-1 protein in the intestine, at Vibrio Enterobacteriaceae Flavobacterium
starvation and 4 hrs after feeding, respectively (Fig. 3C). Cytophaga

A
U mg-1 protein

0,6
100
0,4
80
0,2 60
%

0 40

0 2 4 6 20
time (hrs) 0
1h 2h 4h
stomach pyloric caeca intestine
gelatine caseine lipid starch elastin chitin

Fig. (3C). Average values of amylase measured in the organs of Figs. (4 a, A). Composition (a) and activity of degradation on or-
greater amberjack Seriola dumerilii (Risso) specimens fed with ganic substrates (A) of the microbial community found in the stom-
fresh offals. See Fig. 3A for caption. ach of greater amberjack Seriola dumerilii (Risso).
80 The Open Fish Science Journal, 2009, Volume 2 Caruso et al.

Six hours after feeding, the bacterial flora belonging to


100
Vibrio genus increased in the intestine (Figs. 4b, B), showing
that a selection in the bacterial composition took place to- 80
gether with an intensification of metabolic versatility. At this
60
digestive phase (6 hrs after feeding), the decomposition ac- %
tivity of the microflora increased, concurrently with a de- 40
crease in the amount of native (endogenous) enzymes to 20
level similar to those detected during starvation. This trend
suggested that bacteria were mainly responsible for the late 0

intestine
Pseudomonas

pyloric caeca
digestive phases (6 hrs after feeding), while biochemical

Flavobacterium

stomach
Vibrio +
degradation accounted for the assimilation of organic sub-

spp.
strates already in the early digestive phases, 2- 4 hrs after
feeding.
b a

100
80
100%
60
%

40
80%

20 60%
0
1h 2h 4h 6h 40%

Pseudomonas Micrococcus Aeromonas 20%


Vibrio Enterobacteriaceae Flavobacterium
Cytophaga 0%
stomach pyloric caeca intestine
B
b
gelatin casein Tween 80 starch chitin
100

80
Figs. (5 a, b). Composition (a) and activity of degradation on sub-
60 strates (b) of the microbial community found in the stomach, pylo-
ric caeca and intestine of blackspot seabream Pagellus bogaraveo
%

40 (Brünnich).
20
In the sampled larvae, the increases observed in length
0 (from 1 to 15 mm) and in weight (from 0.07 to 2.48 g) from
1h 2h 4h 6h hatching (0 DAH) to the end of the experiments (45 DAH)
gelatin casein Tween 80 starch elastin chitin were paralleled by morphological differentiation of the di-
gestive tract in oesophagus, stomach, midgut and hindgut
Figs. (4 b, B). Composition (b) and activity of degradation on or- (not shown in Figures).
ganic substrates (B) of the microbial community found in the intes-
tine of greater amberjack Seriola dumerilii (Risso) Qualitative variations in the distribution of digestive en-
zymes were recorded, in relation to the functional differen-
In blackspot seabream (Figs. 5a, b), the qualitative study tiation of the digestive tract. As shown in Fig. (6), from 0 to
of the microbial flora occurring in the different organs of the 17 DAH, the total proteolytic activity prevailed at alkaline
digestive tract showed the highest bacterial densities in the pHs, whilst between 24 and 31 DAH, the percentage of the
pyloric caeca (2.34 x 106 CFU g-1), followed by stomach and acid protease increased, predominating over the alkaline
intestine. Motile and glucose fermenting microorganisms, ones during the final stages (38-45 DAH).
belonging to Vibrio and Pseudomonas genera, were pre- Table 2 reports the enzymatic values detected during the
dominant in the intestine and pyloric caeca, while strains larval growth of common pandora, showing enzymatic levels
belonging to Flavobacterium were detected mostly in the increasing with the age. At day 45 the pepsin content was on
stomach. The microflora found in the pyloric caeca dis- average nine times higher than that of day 3. In the early
played gelatinolytic, lipolytic and amylolytic capabilities; stages, lipase was not detectable, suggesting a poor lipid
strains able to degrade chitin were observed mostly in the metabolism; larvae showed increased ability to hydrolyse
intestine (Figs. 5a, b). lipids from day 31.

Effect of Life Stage Species Diversity


A preliminary investigation of the digestive enzymatic In Epinephalus spp., the enzymatic patterns suggested a
activities was performed during the first 45 days of devel- higher enzyme availability and a more diversified enzyme
opment of common pandora larvae fed live prey. distribution along the digestive tract of grouper than in dusky
Digestive Enzymes in New Teleost Species for Aquaculture The Open Fish Science Journal, 2009, Volume 2 81

0-17 DAH 0 3 6
400 1820 a
U mg-1protein
300 10 17 600
200

U m g protein
100 400

-1
0
200
1 2 3 6 8 9 10

0
400
24-31 DAH 600 b
24 31
U mg-1protein

300

U mg protein
200 400
stomach

-1
100 200 intestine
0
1 2 3 6 8 9 10 0

Am ylase
eptidase

Elastase

Lipase
Total proteases

Leucin
38-45 DAH
400 38 45

Am i
U mg-1 protein

300
200
100 Figs. (7 a, b). Digestive enzymes detected in the stomach and intes-
0 tine of specimens (n=5) of grouper Epinephalus aenaeus (Geoffrey
1 2 3 6 8 9 10 St Hilaire)(a) and dusky grouper E. marginatus (b) (Lowe).
pH

the acquisition of basic knowledge on this field of research.


Fig. (6). pH dependence of proteolytic activity during the larval On the other hand, the determination of digestive enzymes in
development of common pandora Pagellus erythrinus L. (n=100). Sparid fish has a wide interest, due to the importance of cul-

Table 2. Enzyme Values (Mean ± s.d., n=100) Measured During the Larval Development of Common Pandora Pagellus erythrinus L.
Values are Expressed as U mg-1 Protein

Days After
Pepsin Trypsin Chimotrypsin Carboxypep. A Carboxypep.B Amylase Lipase
Hatching

0 0.27 ± 0.00 0.15 ± 0.00 0.19 ± 0.03 0.06 ± 0.00 0.03 ± 0.00 0.04 ± 0.00 0.00 ± 0.00

3 0.34 ± 0.01 0.16 ± 0.00 0.22 ± 0.01 0.06 ± 0.00 0.04 ± 0.01 0.06 ± 0.00 0.00 ± 0.00

6 0.42 ± 0.01 0.17 ± 0.00 0.29 ± 0.01 0.07 ± 0.00 0.04 ± 0.03 0.07 ± 0.00 0.00 ± 0.00

10 0.65 ± 0.00 0.19 ± 0.01 0.32 ± 0.04 0.07 ± 0.00 0.05 ± 0.00 0.15 ± 0.01 0.00 ± 0.00

17 0.68 ± 0.01 0.21 ± 0.05 0.45 ± 0.01 0.08 ± 0.00 0.07 ± 0.00 0.15 ± 0.02 0.00 ± 0.00

24 1.01 ± 0.00 0.21 ± 0.07 0.69 ± 0.01 0.09 ± 0.01 0.09 ± 0.00 0.17 ± 0.02 0.00 ± 0.00

31 2.11 ± 0.07 0.22 ± 0.01 0.72 ± 0.03 0.09 ± 0.00 0.09 ± 0.00 0.17 ± 0.00 7.35 ± 0.45

38 2.35 ± 0.01 0.25 ± 0.02 0.99 ± 0.02 0.13 ± 0.08 0.10 ± 0.01 0.26 ± 0.00 7.35 ± 0.23

45 2.80 ± 0.01 0.34 ± 0.00 1.78 ± 0.02 0.18 ± 0.01 0.11 ± 0.00 1.24 ± 0.03 9.80 ± 0.13

grouper (Figs. 7 a, b). In both the species, the highest levels turing these fish as potential resource for the Mediterranean
of total proteases, leucine aminopeptidase and amylase were market.
detected in the intestine, whereas in dusky grouper elastase
To date, current understanding of digestive patterns in
predominated in the stomach. new potential “candidates” for future diversification of
Mediterranean aquaculture is still highly fragmentary and
DISCUSSION
incomplete (i.e. Diplodus puntazzo [17, 19]; Pagellus spp.
This study is a first attempt to review the information ex- [48-49]). This is in contrast with the wide knowledge avail-
isting on digestive enzymes in some emerging Teleost spe- able on traditionally intensively reared species such as sea
cies for the Mediterranean productions. Data from several bass [50], sea bream [6, 18, 51-53] and rainbow trout [6, 54].
species that are considered of potential interest for fish farm-
This review focuses on particular features concerning the
ing have been collected, with the objective of contributing to
digestive capability of some selected Teleost species, such as
82 The Open Fish Science Journal, 2009, Volume 2 Caruso et al.

those related to diet composition, digestive phases, life with the secretion of specific enzymes [73]. The predomi-
stages, contribution of bacterial enzymes to the decomposi- nance of the acid protease in the late stages (24-31 DAH)
tion of food ingredients, and the species diversity. was not surprising, because sub-mucosal gastric glands pro-
liferated considerably at this stage, allowing a more efficient
Response of Enzymatic Patterns to Dietary Composition digestive process. At the earliest stages of development,
common pandora larvae displayed high levels of trypsin and
Fish may adapt their metabolic functions to the dietary
chymotrypsin; this was similar to what found in other spe-
substrates, through a regulation in enzyme secretion, in order
cies [74]. Trypsin content increased with increasing age, in
to improve the utilisation of feed ingredients. The enzyme
contrast with turbot larvae [75]. The increase observed in
profiles found in axillary seabream fed two different diets
larval enzymatic contents with increasing age agreed with
confirmed this consideration. A similar result was observed
by Hofer [55, 56] and Reimer [4] for proteases and amylases. those reported for other fish species [74, 76] and could be
related to the folding of the intestinal epithelium, that in-
Temporal Variations in Enzymatic Distribution During creased the surface available for absorption during fish
Digestive Process growth [37, 77].

Changes occurring in the secretory response after feeding Species Diversity


may provide useful insights on the evolution of the digestive Results obtained during comparative studies are not abso-
process. Till now, only a few studies have investigated the lute and may not be directly comparable with those reported
changes in enzyme patterns which take during digestion in by other references, due to the broad variety of techniques
some fish species such as carp, Cyprinus carpio [57], rain- and to the different modalities of enzyme extraction (by
bow trout Oncorhynchus mykiss [58], Japanese (Anguilla scraping of the gastrointestinal mucosa or by homogenizing
japonica, [59]) and European (Anguilla anguilla [60]) eel, the whole digestive tract) which cause variability in the final
African catfish Clarias gariepinus [61] and Atlantic salmon values [6]. Nevertheless, some useful considerations for op-
Salmo salar [62]. In greater amberjack, enzymatic activities timising nutritional protocols may be inferred from the
showed a peak 4 hrs after feeding, followed by a decrease at whole data set obtained during our research, schematised in
levels similar to those detected at starvation. A possible ex- Table 3.
planation for this trend is the occurrence of enzymatic de-
naturation or not reversible bound between enzyme and sub- In greater amberjack, the decrease of proteases along an
strate. antero-posterior gradient [25] is consistent with the patterns
recorded in other fish species [78, 79]; this means that gas-
Role of Bacteria In Fish Digestion tric proteolysis contributes largely to the total proteolytic
capability. The result could depend on warm water
Previous studies [63-66] have underlined the importance temperature; in fact, in the related Seriola quinqueradiata
of microbes associated to the gastro-intestinal mucosa as key seasonal changes were found, with higher trypsin and
players in digestive processes. Microbial distribution ob- chymotrypsin activities in the intestinal contents and lower
served in greater amberjack showed how intestinal microbes pepsin activity in the stomach contents at low water
may provide an important contribution to the digestion and temperatures [80]. The low amylase levels measured in the
nutrition of fish, releasing nutrients for intestinal absorption. stomach of greater amberjack are in agreement with that
From the enzymatic profiles detected in amberjack and observed in other carnivorous fish, confirming the existence
blackspot seabream, we concluded, in agreement with Sera of a close relationship between amylase activity and feeding
[67], that bacteria, initially introduced with food, further habits [6, 52].
become adapted to particular gastro-intestinal environmental
conditions, and develop as a distinct even if transient com- Comparison between enzyme activities measured in axil-
munity of facultative aerobic and anaerobic bacteria, which, lary seabream, blackspot seabream and common pandora
being metabolically active, play an active role in digestive [29] revealed that the distribution of digestive enzymes
processes. changed in relation to the specific diversity.
In the juveniles of blackspot seabream the enzymatic
Ontogeny of Digestive Enzymes
contents in the gastrointestinal tract were in the same order
Many recent studies have focused on the determination of magnitude as those previously measured in adult speci-
of digestive enzymes in marine fish larvae; they aimed at mens [22, 29]. In the three Sparid species, digestive enzymes
describing different aspects of the physiology of the larval were measured at different levels, showing significantly (P <
digestive tract such as digestive enzyme profiles as well as 0.01) lowest contents and therefore the lowest digestive abil-
changes occurring in the metabolic processes during the on- ity in blackspot seabream compared to axillary seabream and
togeny of the digestive tract; the relationships between larval common pandora (Table 3). In particular the low levels of
nutrition and development have also been investigated [see proteases found in blackspot seabream would indicate that in
68-70 for reviews on nutritional physiology in larvae and this fish protein digestion is potentially poor. As a conse-
juveniles]. quence, blackspot seabream could require for the digestion a
time longer than similar species, with a longer time of reten-
During the first month of life, the gut of common pan-
tion of digesta along the gastrointestinal tract and a lower
dora larvae underwent morphological changes that are re-
lated to the transition from endo- to exogenous feeding, as growth efficiency. Conversely, in axillary seabream the de-
tection of high levels of proteases, among which particularly
commonly observed in fish species [71, 72]. The regional
leucine aminopeptidase, suggest that this species can easily
differentiation of the digestive tract is generally associated
Digestive Enzymes in New Teleost Species for Aquaculture The Open Fish Science Journal, 2009, Volume 2 83

Table 3. Comparison Between the Enzyme Levels Detected in the Organs of New Candidate Fish Species for Aquaculture. Data are
Reported as U mg-1 Protein

Stomach
Enzyme
S. dumerilii P. acarne P. bogaraveo P. erythrinus E. aenaeus E. marginatus

Tot. proteases 123.0 ± 0.04 430.0 ± 2.7 1.29 ± 0.01 87.0 ± 1.8 337.0 ± 120.0 85.2 ± 15.0

Pepsin nd nd 0.36 ± 0.03 nd nd nd

Trypsin nd nd 0.00 ± 0.00 nd nd nd

Chymotrypsin nd nd 0.00 ± 0.00 nd nd nd

Carboxyp. A nd nd 0.00 ± 0.00 nd nd nd

Carboxyp. B nd nd 0.02 ± 0.01 nd nd nd

Leuc. Aminopep. 53.5 ± 0.33 26.17 ± 1.08 nd 15.43 ± 1.32 95.09 ± 21.0 28.77 ± 14.0

Elastase 0.03 ± 0.00 0.67 ± 0.02 nd 2.50 ± 0.27 139.04 ± 12.0 46.47 ± 15.0

Amylase 0.04 ± 0.00 6.50 ± 0.20 0.09 ± 0.02 34.06 ± 1.47 21.76 ± 0.03 5.33 ± 0.05

Lipase 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Pyloric caeca

Enzyme S. dumerilii P. acarne P. bogaraveo P. erythrinus

Tot. proteases 142.5 ± 0.02 349.5 ± 8.1 7.85 ± 0.006 299.5 ± 3.08

Pepsin nd nd 0.00 ± 0.00 nd

Trypsin nd nd 0.03 ± 0.01 nd

Chymotrypsin nd nd 0.55 ± 0.02 nd

Carboxyp. A nd nd 0.10 ± 0.04 nd

Carboxyp. B nd nd 0.02 ± 0.01 nd

Leuc. Aminopep. 121.00 ± 0.90 66.79 ± 3.40 nd 13.67 ± 2.88

Elastase 2.30 ± 0.02 2.77 ± 0.01 nd 0.54 ± 0.33

Amylase 0.28 ± 0.01 14.07 ± 0.41 0.14 ± 0.03 16.58 ± 0.91

Lipase 3.30 ± 0.01 8.12 ± 1.08 2.0 ± 0.5 9.84 ± 0.21

Intestine

Enzyme S. dumerilii P. acarne P. bogaraveo P. erythrinus E. aenaeus E. marginatus

Tot. proteases 137.5 ± 10.01 352.5 ± 3.1 6.97 ± 0.01 368.5 ± 4.1 1819.0 ± 12.0 477.6 ± 34.0

Pepsin nd nd 0.00 ± 0.00 nd nd nd

Trypsin nd nd 0.07 ± 0.01 nd nd nd

Chymotrypsin nd nd 0.07 ± 0.01 nd nd nd

Carboxyp. A nd nd 0.08 ± 0.06 nd nd nd

Carboxyp. B nd nd 0.03 ± 0.01 nd nd nd

Leuc. Aminopep. 99.00 ± 0.90 29.02 ± 0.91 nd 25.18 ± 1.44 166.03 ± 4.0 34.27 ± 21.0

Elastase 0.65 ± 0.01 2.06 ± 0.03 nd 1.41 ± 0.16 200.56 ± 13.0 22.12 ± 6.0

Amylase 0.18 ± 0.01 12.46 ± 0.50 0.10 ± 0.03 20.05 ± 1.60 31.95 ± 0.06 7.88 ± 0.03

Lipase 5.35 ± 0.09 9.89 ± 2.31 0.40 ± 0.10 12.53 ± 0.28 18.2 ± 0.05 7.5 ± 0.03
84 The Open Fish Science Journal, 2009, Volume 2 Caruso et al.

grow on diets rich in proteins. In grouper, the high levels of them potentially able to digest, absorb and metabolise en-
proteases compared to the other examined species (Table 3) dogenous food of different biochemical composition, inde-
confirmed the great ability of this species to digest dietary pendent from live prey ingestion. Consequently, exogenous
proteins. proteases from live food (rotifers) are supposed to contribute
scarcely to the digestive process, in disagreement with what
Concerning the enzyme distribution along the digestive
previously observed in other fish larvae [74, 75]. The basic
tract, while in axyllary seabream a large fraction of prote-
evaluation of the functional status of larval fish gut repre-
olytic activity was found in the stomach, in blackspot se-
abream, as well as in common pandora, protease levels were sents the initial step for defining the optimal rearing condi-
tions of this species [28]. Due to the increasing tendency to
higher in the intestine than in the other organs [29]. A similar
replace live food with inert microdiets, knowledge of larval
enzymatic distribution was detected along the digestive tract
digestive ability of this species and of the qualitative and
of Sparids (sharpsnout seabream Diplodus puntazzo (Cetti)
quantitative variations in the distribution of digestive en-
[17]. Amylase and leucine aminopeptidase were measured in
zymes during the first development stages is likely to im-
higher amounts in the pyloric caeca of axillary seabream and
blackspot seabream, while lipase was detected at the highest prove the performances of larval rearing and to contribute to
weaning success of common pandora.
levels of in the intestine of axillary seabream and common
pandora.
CONCLUSIONS
Practical Application of Enzymatic Data for Setting Up Research on the metabolic processes related to digestion
Feeding Protocols in reared fish is still in progress and, in those species cur-
rently farmed, the status of research on digestive physiology
Knowledge of the digestive capability of reared species is
is far from a complete picture on the process of nutrient hy-
necessary for adapting dietary formulation to the functional-
drolysis. Therefore, further investigations on digestive en-
ity of the digestive tract and represents one of the most im-
zymes are required to improve knowledge existing on their
portant objectives of research in fish nutrition [25]. As diges-
interaction with different factors intrinsic to fish nutrition
tive enzymes can be used as surrogate parameters to predict
the ability of fish to use different nutrients, in vitro meas- (such as dietary composition or growth stage); all these fea-
tures can offer interesting perspectives for further studies,
urements of digestive enzymatic activity may allow to adapt
with exciting and promising applicative purposes for aquac-
artificial feeding to the nutritional needs of fish [54].
ulture development. The clarification of aspects intrinsic to
In greater amberjack, the detection of low amylase levels the digestive physiology, such as the definition of the enzy-
suggests that carbohydrate digestion is not so efficient; such matic pattern typical of a selected fish species, the chronobi-
a consideration may be helpful for the production of manu- ology of the digestion and the evolution of the digestive or-
factured, dried feeds specific for greater amberjack. Con- gans during fish growth can provide useful contributions to
versely, the amylase equipment found in some Pagellus spe- the field of fish nutrition.
cies such as P. erythrinus (Table 3) would represent for
those species a good strategy for utilising diversified feeding ACKNOWLEDGMENTS
resources, as previously observed [48].
Research was funded by Italian Ministry for Agriculture
As an example of potential utilisation of the enzymatic and Forest Policies (MIPAF) within the 5th Three-years plan
data to optimise feeding practices, from values obtained in of research and experimentation.
seabream species some suggestions about the choice of both
time intervals and nature (fresh or dried) of food adminis- REFERENCES
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Received: June 13, 2008 Revised: February 06, 2009 Accepted: February 09, 2009

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