Camp. Biochem. Physiol. Vol. 85A, No. 3, pp.

545-551, 1986
Printed

in Great

0300-9629186

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ACTIVITIES OF CHITINASE AND PROTEASE AND

CONCENTRATION
OF FLUORIDE
IN THE DIGESTIVE
TRACT OF ANTARCTIC
FISHES FEEDING ON
KRILL (EUPHA USIA SUPERBA
DANA)
H. REHBEIN,* E. DANULAT~ and M. LEINEMAN*
fiir Fischerei, Institut fiir Biochemie und Technologie, Palmaille 9, 2000
Hamburg 50, FRG. Telephone: (040) 38905-l 19
TInstitut fur Hydrobiologie und Fischereiwissenschaft, Zeiseweg 9, 2000 Hamburg 50 FRG

*Bundesforschungsanstalt

(Received 20 February 1986)

Studies on the digestion of krill by Notothenia rossii marmorata, Notothenia neglecta,
Champsocephalus gunnari and Chaenocephalus aceratus showed that these Antarctic fish species are well

Abstract-l.

equipped to feed on krill, as indicated by their high levels of chitinase and protease activity.
2. Very high chitinolytic activities were determined in the stomach of the fish species. However,
activities that were measured in intestine samples can be substantial, as well. Very strong protease activities
were determined in samples of the stomach tissue and the intestinal contents.
3. When krill were present in the guts, the concentrations of fluoride in the stomach and intestinal
contents of N. rossii marmorata and Ch. gunnari were extremely high, while the tissues were practically
devoid of fluoride.

INTRODUCTION
Anderson et al. (1978) reported the total weight of the
population of krill, Euphausia superba, to be approximately 3 billion tons in Antarctic waters, where they
are the main food organism for secondary consumers.
According to the estimate of Kock (1985a), several
million tons of krill are consumed by Antarctic
Notothenioid fishes per year in western Antarctic
waters.
Like all crustaceans and insects, Euphausiu superba
has a chitinous exoskeleton. Chitin accounts for
3.2% of whole krill (Anderson et al., 1978). Furthermore, the exoskeleton of Euphausia superba normally
contains up to 3000-4260mg fluoride per kg dry
weight (Soevik and Braekkan, 1979). One way in
which the fish might avoid the fluoride burden is the
excretion of undigested shells. On the other hand, the
chitin in the shells could be an energy source for fish
that may become accessible by enzymatic hydrolysis
(Alliot, 1967; Peres et al., 1973; Lindsay et al., 1984;
Danulat, 1986a). The biological decomposition of
chitin is carried out by a system of exocellular
enzymes, known as chitinases (EC 3.2.1.14) and
chitobiases (EC 3.2.1.29), which consecutively hydrolyse the polysaccharide to the monomer j-N-acetylD-glucosamine (Jeuniaux, 1966). While chitinase activity has been reported in numerous fish species
living in temperate and tropical waters (Jeuniaux,
1961; Okutani, 1966; Sera and Okutani, 1968; Micha
et al., 1973; Goodrich and Morita, 1977a,b; Danulat
and Kausch, 1984; Lindsay, 1984), this paper
presents the first report on the occurrence of chitinase
in the guts of Antarctic fish.
To gain some information about the mode of
digestion of krill by Antarctic fish and the fate of
fluoride from krill shells, chitinase and protease activ-

ities and fluoride concentrations in the guts were

determined and correlated with the feed of the fish.
MATERIALS AND

METHODS

Fish material

Fish samples were collected during the first part of the

German Antarctic Expedition 1985 (Kock, 1985b) from
January to March 1985) (68th cruise of FRV Walrher
Herwig).
Notothenia rossii marmorata was caught east off South
Georgia, Notothenia neglecta near Elephant Island and the
icefishes Champsocephalus gunnari and Chaenocephalus
aceratus in the waters around the South Orkneys. Table 1

shows the catch data and some biological parameters of the

fish. As soon as they were on board, the fish were identified
(Norman, 1938) measured and dissected. Stomachs and
intestines were emptied, contents and tissues were frozen
separately in plastic bags by a stream of cold air (- 40C).
The deep frozen samples were stored for 6 months at - 25C
until being analysed in the laboratory.
Preparation of extracts

Thawed tissues were washed using ice-cold distilled water

and blotted on filter paper. The wet weights of the entire
samples were determined. After dilution 1:2 (w/v) with
distilled water, the samples were homogenized by means of
an Ultra Turrax (Janke & Kunkel, FRG). The enzymes were
extracted at 0C for 24 hr. The following day, centrifugation
was carried out at 48,000g max for 20min at 5C. The
supematants, referred to as the enzyme solutions or
extracts, were tested for chitinase and protease activities
as outlined below.
Chitinase assay

The end product measurement of Jeuniaux (1966) was

used to determine chitinase activity in the enzyme extracts.
The procedure was modified in that 4ml of test mixture
contained 100 ~1 of toluene; B-glucosidase (Sigma G 8625)
in a concentration of 1.5 units per ml distilled water was
545

H. REHBEIN et nl.

546
Table

I. Fish samples;

NO.

Fish species

all specimen of the respective fish species came from one haul
Fish
length
(cm)

Oriein
Fish
Sex*

II85 Notorhenia rossii

II86
marmorata
1187
1188
1189

43
48
49
43
46

1222 Champsocepholus gunnari

1223
1224
1225
I226

40
38
42
43
41

1235 Chaenocephalus acerafus

1236
1237
1238
1239

n.d.t
n.d.
n.d.
n.d.
n.d.

n.d.
rd.
n.d.
n.d.
rd.

40

1391 Notothenia negkcta

m
f
m

of the samples

Position

Date

(catch data)
Depth

(m)

5K 33.5 s
35 36.1 W

8.2.85

240-251

60 31.0S
45 11.4 w

14.2.85

255-282

61 6.4 S
46 28.7 W

16.2.85

273-286

60 56.3 S
55 36.4 W

25.2.85

7&9l

m
m
m
m

*m = male; f = female.
tn.d. = not determined.

used. Prior to use, a suspension of 5 mg purified, native

chitin per ml (Serva 16620) incl. 1% toluene was stirred on
a magnetic stirrer for 48 hr. In the two controls, distilled
water was substituted for the chitin suspension (control I)
or the enzyme solution (control
II).
After incubation
for 180 min at 37C. the N-acetylD-glucosamine
assay by Reissig ef al. (1955) was employed,
using 1000 ~1 supernatant
and doubled volumes of reagents.
By increasing
the concentration
of p-dimethylaminobenzaldehyde
in the reagent by 50%, it was possible to
widen the range in which optical density (OD) was proportional to the amount
of amino-sugar.
After cooling for
5 min in an icebath, the samples were read at 585 nm against
the reagent blank.
QuantSfication. A standard curve was used to convert OD
readings to pg N-acetyl-D-glucosamine
(NAG). The points
were determined
in duplicate.
Correlation
coefficient
is
0.9984, m = 0.1294, b = 0.0064. Control readings were subtracted from sample readings.
Result are reported
as pg
NAG per hr per g wet weight of sample or pg NAG per hr
per mg protein.
Protease assays and protein content
Protease activities were measured
using hemoglobin
as
substrate (Anson, 1939). Stomach tissue and contents were
tested for acid protease activity (Fgnge and Grove, 1979):
400~1 of 1% (w/v) hemoglobin
(Merck,
Darmstadtprotease substrate according
to Anson) dissolved in 0.5 M
sodium
acetate
pH 3.0 was mixed with 100~1 enzyme
solution and incubated at 37C for 30 min. The reaction was
stopped by addition
of 500~1 of ice-cold 10% (w/v) trichloracetic acid. The precipitation
of protein was completed
by placing the test mixture into ice-cold water for 30min,
followed by centrifugation
at 8000 g for 1.5 min (Eppendorf
centrifuge 3200). A 400 ~1 volume of supernatant
was mixed
with 200~1 of diluted Folin Ciocalteus
Phenol Reagent
(Merck; diluted 1: 2, v/v. with distilled water) and 400 ~1
1 M NaOH. After 5-lOmin, the OD 578 was read against
a blank. The blank was prepared
adding first TCA, then
enzyme solution to the assay mixture.
Intestinal tissue and contents were tested for pancreatic
and intestinal protease activity at pH 7.5 (Flnge and Grove,
1979). Hemoglobin
(2 g per 100 ml) was dissolved in a
solution containing 6 M urea and 10 mM sodium potassium
phosphate,
pH 7.5.
A 200 ~1 volume of hemoglobin
solution were mixed with
100~1 enzyme solution and incubated
at 37C for 30min.

The reaction was then stopped by addition of 700 ~1 ice-cold

10% TCA. From this point, the procedure described above
was followed
but the optical density was measured
at
750 nm.
Tyrosine was used as standard,
and protease activities
were calculated as pg tyrosine per pg protein per hr. or pg
tyrosin per hr per g wet weight of sample. In most cases, the
extracts had to be diluted with distilled water,
Protein content was determined
with the BCA Protein
Assay Reagent (Pierce, Rockford, Illinois), applying the
micro protocol and using bovine serum albumin as protein
standard
(Pierce, 1984).
Determination ofjuoride

contenf

A 1 g portion

of the sample homogenate

prepared
by
means of an Ultra Turrax as described above was extracted
with 0.1 M HNO, (2 x 25 ml), 50 ml buffer was added and
the fluoride concentration
of the resulting acidic solution
was determined
potentiometrically
by means of a fluoride
sensitive electrode.
The method was given by Alusuisse (1976).
Determination of pH value
The pH was measured
combination
electrode.

in the sample homogenate

using a

RESULTS AND DISCUSSION

Examination
of the digestive tracts of N. rossii
and Ch. gunnari revealed that the fish had
been feeding on krill (Tables 2 and 3). The stomachs
of Ch. aceratus were empty, whereas the inspection of
the intestinal contents showed that one specimen had
eaten krill a while before catch (Table 4).
Macroscopic
examination
of gut contents showed
the degradation
of the krill exoskeleton proceeded in
the digestive tract. Results presented in Tables 2-4
indicated that these Antarctic fish species are well
equipped to digest krill due to marked activities of
chitinase and protease in the digestive tract.
Tests on chitinase
activity included
the determination
of the original
content
of N-acetylD-glucosamine
in the samples, being described as
control I of the chitinase assay (see Materials and
Methods).
In tissue extracts, no detectable levels of
marmorata

(SD,

*Sample code: SC = stomach

content,

ST = stomach

tissue, IC = intestine

,)

Greenish liquid with

grey-white particles,
shells not detectable

Krill; pale rosecoloured specimen,

not digested

1189

deviation

Dark liquid containing

small residues of
shells

Krill; undigested pale

rose coloured specimen
in a colourless liquid

II88

Mean value k standard

Deep red liquid

containing grey-white
particles, shells not
detectable

Krill; red liquid with

white muscle particles,
black eyes, shells not
detectable

1187

1186

1185

Mainly reddish-brown
residues of shells,
small volume of liquid

Intestine

Krill; identifiable as
entire specimen, exoskeleton
was thin and soft,
sticking on the muscle

content

Clear, light liquid

with grey-white
particles

Stomach

Krill; heavily digested,

exoskeleton of the
cephalothorax
identifiable
colour: light

Fish
NO.

Kind of food and stage of its digestion

content,

IT = intestine

IT
tissue.

6.7
kO.2

8.5
kO.4

6.5
io.1

ST
IC

6.4
* I.2

7.4
6.6
8.4
6.7

7.0
6.5
8.8
6.7

4.5
6.3
7.9
6.4

SC

SC
ST
IC
IT

SC
ST
TC
IT

SC
ST
IC
IT

7.0
6.6
8.4
6.9

6.3
6.6
8.9
6.8

SC
ST
IC
IT
SC
ST
IC
IT

pH value

Sample

Table 2. Nofofhenia

4.0
i2.0

424
k 572

5.7
k3.7

388
f 332

157
2.5
I2
0.8

I48
3.9
200
4.0

570
I2
220
3.8

890
4.5
1435
6.1

175
5.7
255
5.2

kg wet wt

mg

Fluoride
content

rossii mormoroto

625.5
1804.0
50.8
66.7
587.2
92.3
114.6
73. I

x.7
14.9
2.1
12.3

168
* I51
88
*79

12.1
f2.9

1457
f 846

15.5
fr I.6
3.1
* I.8

479
&I66

9.3
k2.8

8.8
13.2
I.5
7.8

245.5
1986.0
162.5
15.7

104,529
* 110,444
11,378
f 5245

2.6
f 2.2

161,114
f 76,045

27.6
+ 15.5
16.9
f 7.4

9742
* 7591

18.3
-7.0

2826
249,435
64,032
4131

22.3
2.3
25.3
3.1

+llz

304

12,412
f9925

3790
*I944

436
+509

107
6299
14,229
177

120
5172
12,710
403

3129
231,189
80,070
14,871
23.9
40.4
8.1
I.8

242
3514
2391
417

8766
134,712
43,029
15,621

389
2366
27.743
311

1323
I599
4985
205

mg protein
x hr

pg Tyrosine

activitv

21,042
72,624
35,895
7446

g wet wt
x hr

pg Tyrosine

Protease

12,948
I 17,609
299,619
14,823

7.4
39.9
14.9
0.3

15.0
28.9
23.8
6.0

574.4
2 190.0
427.6
223.2

mg protein
x hr

NAG

22.9
26.6
12.2
1.7

hr

Irg

activity

363.7
1213.0
85.9
60.3

g wetwt

pg NAG

Chitinase

II.1
16.6
3.6
15.9

12.8
17.4
6.0
12.5

5.3
15.2
2.4
12.1

ml extract

mg

Protein
content

content,

ST = stomach

,)

tissue, IC = intestine

content,

344
5.8
213
2.2

6.9
6.4
8.4
6.6
306
*74
7.1
k4.l
488
+240
5.1
f 5.3

292
4.6
804
2.6

7.4
6.7
8.8
6.5

7.0
f0.3
6.4
f0.2
8.5
kO.2
6.6
f0.2

263
14.3
323
n.d.t

220
4.5
450
I3

7.1
6.3
8.4
6.4
7.1
6.4
8.6
6.8

410
6.5
650
2.7

tissue

kg
wet wt

6.6
6.4
8.3
6.6

PH
value

gunnari

Fluoride

3. Champsocephnlus

IT = intestine

IT

IC

ST

SC

SC
ST
IC
IT

Liquid without shells

containing grey-white
particles

Krill; as 1222

(SD,

SC
ST
IC
IT

Small amount of IC,

mainly consisting of
shells

Krill; as 1222

deviation

SC
ST
IC
IT

Reddish-brown
liquid
with a large amount of
shells

Krill; as 1222

Mean value k standard

SC
ST
IC
IT

Deep red liquid

containing many
shell particles

Krill; as 1222

content
SC
ST
IC
IT

Intestine

Deep red liquid

containing many shell
particles

content

Krill; stomach was

tightly filled with
undigested krill

Stomach

Sample code: SC = stomach

tn.d. = not determined.

1226

1225

I224

I223

I222

Fish
NO.

Kind of food and stage of its digestion

Table

18.3
f4.3
14.7
+3.0
4.8
f 1.2
16.7
+4.8

12.5
12.3
4.3
15.2

22.4
15.1
3.1
15.0

15.2
18.3
5.0
23.6

19.4
II.1
6.2
18.8

21.8
16.6
5.4
IO.8

ml extract

Protein
content
mg

activity

333
?60
1090
+ 329
200
20
208
k8l

265
734
I75
I75

428
I232
I82
344

332
1414
214
I98

309
740
207
IO1

329
1331
220
I31

wet wt
x hr

Chitinase

6.2
+ 1.0
24.4
f 3.2
14.4
+3.1
4.3
+ 1.9

7.1
19.9
13.6
3.8

6.4
27.2
19.5
7.7

7.3
25.8
14.3
2.8

5.3
22.2
11.1
3.4

5.0
26.7
13.6
4.0

mg protein
x hr

pg NAG

activity

2168
+850
5 I ,430
f 24,787
154,714
k35.190
33,602
+ 12,582

206 I
18,630
133,044
14,046

2826
43,671
109,932
49,182

3207
83,832
185,532
35,382

II46
44,589
193,671
33,582

1602
66,426
151,389
35.8 I7

g wet wt
x hr

peg Tyrosine

Protease

42
*21
II34
+407
10,852
*I224
720
+36l

55
505
10,313
308

42
964
11,821
1093

70
1527
12,369
500

20
I339
10,412
595

25
1334
9345
II05

g wet wt
x hr

pg Tyrosine

Without content

Residues of food
not detectable, milky
light liquid containing
particles of fish
bones

Without content

Without content

1236

1237

1238

I239

*Sample code: SC = stomach

tn.d. = not determined.

content,

ST = stomach

Mean value k standard deviation (SD,

Without content

Stomach content

tissue, IC = intestine

,)

Small content of
light liquid

Dark grey liquid

containing residues
of fish bones

Light liquid with one

fish bone
intestine tissue:
mucosa was liquified

Without content

Particles consisting of
residual krill shells
and grey-green
coagulum

Intestine content

Kind of food and stage of its digestion

1235

Fish
NO.

content,

IT = intestine

IT

IC

SC
ST

SC
ST
IC
IT

SC
ST
IC
IT

SC
ST
IC
IT

SC
ST
IC
IT

SC
ST
IC
IT

Samole

3.0

tissue.

6.5
kO.2
7.9
kO.6
6.6
f0.2

6.8
7.2
6.7

6.4
8.6
6.8

I.1
f0.4
58
*95
3.3
f3.0

1.0
2.0
1.0

1.0
25
2.0

7.0
1.5
6.0
2.0

6.4
3.2
6.2
7.8
6.6

0.5

1.5
200
8.6

kg wet wt

mg

6.5

6.5
8.1
6.7

PH
value

Fluoride
content

Protein

14.6
+ 3.6
4.6
f 3.2
17.2
+ I.6

10.4
a.2
n.d.

16.0
0.5
17.0

II44
+ 429
195
f 345
220
f266

1092
21.6
n.d.

1095
25.2
61.8

47.7
610
21.9
76.2

III

16.4
9.9
11.2
4.9
15.9

1810

1111
712
73.2

26. I
+7.1
17.2
f23.4
1.6
^ _
*tJ.>

35.0
0.89
n.d.

22.8
15.8
I .2

1.6
18.1
1.5
I.6

2.3

32. I

22.6
50.8
1.3

g wetwt
x hr

pg NAG
mg protein
x hr

pg NAG

Chitinase activity

18.8

16.4
4.7
19.5

ml extract

content

197,060
+ 83,426
220,220
f 19,845
25,240
f 17,341

n.d.
n.d.
n.d.

195,762
n.d.t
n.d.

11,757
102,462
234,252
44,616

11,178

305,553

I 84,46 I
206,187
19.926

x hr

g wetwt

pg Tyrosine

4074
f994
15,280
f 928
501
f380

n.d.
n.d.
n.d.

4078
n.d.
n.d.

396
3049
15,936
935

5418

3749
14,623
341

mg protein
x hr

/Ig Tyrosine

Protease activity

H.

550

REHBEIN et al.

NAG were established. However, per g wet wt of

stomach contents of N. rossii marmorata and Ch.
gunnari, up to 2000 pg NAG were determined, while
extracts of their intestinal contents contained up to
3OOOpg NAG per g wet weight.
In the following, the distribution of enzyme activities and the relationship between gut contents and
fluoride concentrations in the different parts of the
digestive tract are presented in detail.
Notothenia

rossii marmorata

(Table 2)

The guts contained krill in all stages of digestion.

The fluoride concentration of the stomach and intestine content of these species differed considerably and
ranged from 175 to 880 mg/kg wet wt in the stomach
content and from 12 to 1435mg/kg wet wt in the
intestine content. The measured fluoride concentration did not always depend on the content of shells
found in the samples. The tissues were nearly devoid
of fluoride (about 4mg/kg wet wt).
Chitinase activity in extracts of the stomach tissue
reached up to 219Opg NAG per hr per g wet wt.
Average activity in the intestinal tissue made up
approximately 6% of the mean activity in the stomach tissue.
In the stomach tissue, acid protease activity was
very high. Presumably, pepsinogen had been converted to pepsin during a 24 hr extraction period
(Finge and Grove, 1979). The intestinal contents
were also characterized by high protease activity
whereas the intestinal tissue contained relatively low
levels of protease activity.
The pH values of intestinal contents and tissue
were within the expected scope, but those of stomach
contents and stomach tissue exhibited surprisingly
high values. This may be caused by buffering the
acidic gastric fluid, regularly found in the stomach of
fishes (Fange and Grove, 1979), during frozen storage
of the samples and preparation of the extract. When
measured on board using samples prepared from
freshly killed fish that had also fed on krill, the
pH-values of gastric and intestinal fluid of Notothenia
neglecta were 3.5 f 0.8 (N = 9) and 7.5 & 0.1 (N = 5),
respectively.
Notothenia neglecta
The chitinase activity (pg NAG/hr per g wet wt) in
the samples from one specimen of the related species
N. neglecta was 163 (stomach content), 1718 (stomach tissue), 121 (intestinal content) and 134 (intestinal
tissue); this fish had a small amount of a milky liquid
in the stomach, whereas the intestinal content consisted of a dark reddish-brown liquid with residues of
shells.
Champsocephalus gunnari (Table 3)
All specimens of this species gave very similar
results. In contrast to N. rossii marmorata, only
undigested krill was found in the stomach of Ch.
therefore no significant
gunnari and probably
differences in the fluoride concentration of all stomach contents of this species (306 + 74 mg/kg wet wt)
could be detected. The same fluoride concentration
was also found in raw krill (Christians et al., 1982).
The intestine contained liquified krill as well as shells.
The fluoride content ranged from 2 13 to 804 mg/kg

wet wt. Again, the tissues contained only 5-7 mg/kg

fluoride. Nearly the same fluoride concentration,
4.3 mg/kg wet wt for stomach and 4.5 mg/kg wet wt
for the gastric tract, have been reported by Oehlenschllger and Manthey (1982). The fluoride concentration in stomach and intestinal tissues was only
slightly higher than in muscle (1.9 mg/kg wet wt) of
Ch. gunnari (Oehlenschlager and Manthey, 1982).
As for N. rossii marmorata, highest chitinase activities were determined in extracts of stomach tissue;
however, the activities in the intestinal tissue also
appeared to be substantial (app. 20% of the average
chitinolytic activity in the stomach tissue).
Chaenocephalus

aceratus (Table 4)

While the stomachs of all specimen of this species

were empty, chitinase activities in extracts of the
stomach tissue were in the same order as described
for N. rossii marmorata and Ch. gunnari, indicating
that chitinase production is independent of the presence of substrate. One specimen (fish No. 1235)
contained krill shells in its intestine. This fish exhibited high fluoride concentration (200 mg/kg wet
wt) as well as chitinase activity (712pg NAG/hr
per g wet wt). The other specimen which had not
fed on krill had very low fluoride concentrations
(2-25 mg/kg wet wt) and chitinase activity (app.
25 pg NAG/hr per g wet wt) in the intestinal
contents.
Ch. aceratzu had very high protease activities
(about 200,000 pg tyrosine/hr per g) in stomach tissue and intestinal contents. In the case of fish No.
1237, the pH value dependence of protease activity of
both organs was investigated. Stomach tissue exhibited no activity when analyzed at pH 7.5, whereas
the protease activity of intestinal contents and tissue
at pH 3.0 was 3.5% and 46% of the respective
activities measured at pH 7.5.
CONCLUSIONS

Compared to chitinase activities in the digestive

tract of many other fish species, in the present study,
very high activities were determined. Considering the
fact that gut samples had been frozen for 3-5 months,
the possibility of loss of some activity has to be taken
into account (Danulat and Kausch, 1984). Results of
all three species confirm earlier findings that in fish
the main site of chitinase production is the stomach
(Okutani, 1966; Micha et al., 1973; Danulat and
Kausch, 1984). However, also chitinase activities in
other parts of the digestive tract may be substantial
(Danulat, 1986a), as in this study the ones measured
in intestine samples, specially of Ch. gunnari.
The presence of high chitinase activities in
stomachs of Ch. acerntus which were free of contents,
confirms earlier findings that chininases are constitutive enzymes in fish (Jeuniaux, 1966; Micha et al.,
1973; Danulat, 1986a,b) and not inducible enzymes
of bacterial origin as reported by Goodrich and
Morita (1977a) for chitinase in the stomach of
Enophrys bison.

Further studies are required to elucidate the role of

chitinases, especially with respect to the very low in
uiuo temperatures in the guts of Antarctic fish. Preferably, future investigations should be based on fresh

Fish feedin ig on krill

fish material or frozen enzyme extracts prepared from

freshly caught fish.
The combined action of protease and chitinase is
necessary for the complete digestion of krill. The
analysis of the gut contents did not allow to draw
clear conclusions about the spatial and temporal
course of digestion; in most specimens of C/r. gunnari
shells have been found in the intestine contents,
whereas other fishes (fish No. 1226, 1185 and 1187)
contained particles of krill flesh in the intestine
contents.
The high fluoride concentration in the gut content
of those fish having no visible shell residues in their
stomachs (N. rossii marmorata, No. 1187) or intestines (Ch. gunnari, No. 1226) indicates that fluoride
must have been released from shells in course of
digestion of the krill. Nevertheless, the low fluoride
content of the stomach and intestine tissues of all
investigated species indicated also that most of the
released fluoride is excreted and not absorbed by the
fish.
Acknowledgements-We
wish
Kundiger for skillful technical

to thank
assistance.

B. Buro

and

R.

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