Antifreeze activity in the gastrointestinal fluids of Arctogadus glacialis (Peters 1874) is dependent on food type
1 University of Tromsø, Norwegian College of Fishery Science, N-9037
Tromsø, Norway
2 Roskilde University, Department of Life Sciences and Chemistry, PO Box
260, DK-4000 Roskilde, Denmark
* Author for correspondence (e-mail: kim.praebel{at}nfh.uit.no)
Accepted 26 April 2005
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Summary |
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Key words: Arctogadus glacialis, Boreogadus saida, antifreeze glycoproteins, freezing avoidance, osmolality, ion concentrations, gastrointestinal fluids
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Introduction |
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Although AFGP comprises 34% (w/v) of the blood in polar fishes
(Ahlgren et al., 1988), and is,
thereby, energetically costly to maintain, no mechanism has been identified
that prevents rectal loss of AFGPs in the Antarctic notothenioids
(O'Grady et al., 1983
).
However, studies of temperate fishes have shown that the fish rectum has the
capacity to absorb small amounts of intact proteins and peptides with
different molecular size and shape (<40 kDa)
(McLean and Ash, 1987
;
McLean et al., 1999
).
The aim of this study was to determine the effect of the ingested type of food on the osmolality, ion concentrations, antifreeze activity and AFGP distribution of the fluids in the gastrointestinal tract of the Arctic gadoid Arctogadus glacialis. Additionally, the hypothesis of AFGP absorption in the intestine of A. glacialis is discussed.
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Materials and methods |
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The specimens were anaesthetized by 30 mg l-1 MS-222 (Sigma Chemical Co., St Louis, MO, USA) and blood was collected from the caudal vein by syringe using a 19-gauge needle. Samples were allowed to clot at approximately 20°C, and thereafter stored overnight at 4°C before centrifugation. After centrifugation at 4000 g for 10 min the serum was removed by pipette and stored at 25°C for later analysis.
The intestinal tract of the dead fish was dissected out, and the oesophagus and rectal end of the tract clamped. The tract was then separated into a stomach, mid-gut and hind-gut portion by clamping between the segments.
Stomach fluid was collected by syringe using a 16-gauge needle puncturing through the stomach wall. The mid-gut and hind-gut fluids were collected by draining the content into 1.9 ml Eppendorf tubes. The fluids were then centrifuged at 4000 g for 10 min and the supernatant was removed by pipette and stored at 25°C.
Observed food content in the stomach was correlated with the colour of the supernatant. Green samples indicated Boreogadus saida (Polar cod) was ingested whereas red samples indicated that crustaceans (Amphipods and Copepods) had been the prey.
Ion concentrations, osmolality and melting-point and freezing-point determinations
Cation concentrations in the body fluids were determined as triplicates
with a FLM3 Flame Photometer (Radiometer, Copenhagen, Denmark). Chloride
concentrations were measured as triplicates with a CMT 10, Chloride Titrator
(Radiometer, Copenhagen, Denmark). Osmolality in the various body fluids was
measured as duplicates with a Wescor 5100C vapor pressure osmometer (Wescor
Inc., Logan, UT, USA).
The freezing point of the body fluids was measured using a Clifton Nanolitre Osmometer (Clifton Technical Physics, Hartford, NY, USA), mounted on a Zeiss STEMI SV11 APO microscope (Carl Zeiss AG, Oberkochen, Germany. The samples were loaded with a capillary micropipette into the center of oil-filled wells and sample size was approximately a third of the well size. The samples were then quickly cooled to 40°C and the temperature was slowly raised until the last ice crystal disappeared, which was taken as the observed melting point. After refreezing the samples, the temperature was slowly raised again until approximately 0.09°C lower than the melting point. The temperature was then lowered 0.19°C and allowed to stabilize for 1 min. Further temperature decrease was then performed at a rate of 0.19°C min-1 until explosive growth of ice spicules occurred. The temperature at which the spicular growth occurred is the hysteresis freezing point, and the temperature difference between the melting point and the hysteresis freezing point is defined as the antifreeze activity.
The measured melting and hysteresis freezing points were given in mOsm, and
by multiplying the osmolality values by 0.001858°C mosmol-1
kg-1 (Levine,
1995), the corresponding temperature (°C) was found. The
maximal thermal hysteresis was then determined as described by Sørensen
and Ramløv (2001
).
Statistical significance was tested at the P
0.05 significance
level by using a two-tailed Student's t-test. Data are given as means
± S.E.M. (N=36).
Electrophoresis
The antifreeze glycoproteins were isolated from the blood and
gastrointestinal fluids by adding cold trichloroacetic acid (TCA; Merck,
Germany) up to a final concentration of 5% (v/v) [antifreeze glycoproteins are
soluble in TCA (DeVries and Wohlschlag,
1969; Van Voorhies et al.,
1978
)]. After centrifugation at 9500 g for 5 min,
the supernatant was transferred to a Spectrapor-3 dialysis tubing (molecular
mass cut off 3.5 kDa; Spectrum Laboratories Inc., Rancho Dominguez, CA, USA),
dialyzed against distilled water at 4°C for 36 h and then lyophilized. The
AFGP were then resuspended in distilled water and fluorescently labelled with
fluorescamine (Sigma Chemical Co.) (Chen et
al., 1997
) and run on 1020% non-denaturing acrylamide
gradient gel using borate in the buffer system
(O'Grady et al., 1982b
), for 1
h at 200 V and 4 h at 30 mA in a cold room. Purified AFGP from the Antarctic
notothenioid Dissostichus mawsoni (kindly provided by A. L. DeVries)
was used as standard. All samples were loaded on the gel in the same total
concentration (250 µg). The gel was viewed with transmitted UV-light and
images captured with an Eagle-eye imaging system (Stratagene, La Jolla, CA,
USA).
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Results |
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The food type had an influence only on the antifreeze activity of the mid-gut fluids. Arctogadus glacialis that had eaten B. saida showed significantly higher hysteresis (antifreeze activity) (2.04±0.31°C) than specimens feeding on crustaceans (1.17±0.08°C) (Fig. 1 and Table 1). The hysteresis freezing points [3.27±0.30°C (Bs) and 2.44±0.11°C (Cr)] for both food types were significantly lower than that of serum (1.99±0.07°C).
The osmolality of the mid-gut fluids was not significantly different from the stomach fluids for the two types of food, but the ion concentrations in the mid-gut fluids were significantly lower than in the stomach fluid, showing the absorption of ions in the mid-gut.
Neither ion concentrations, osmolality nor antifreeze activity of the hind-gut fluids showed significant differences for the two food types. Nevertheless the hysteresis freezing points were significant lower (Bs: 2.74±0.19°C, Cr: 2.53±0.20°C) than the hysteresis freezing point of the serum (1.99±0.07°C) (Fig. 1 and Table 1).
Gel electrophoresis
Native gel electrophoresis revealed that the stomach fluids and the
intestinal fluids contained AFGP in all the different size groups
(Fig. 2). A distinctive smear
is seen below each known AFGP-size showing that the AFGPs are degraded into
many sub-sizes (>40) during the digestion process.
|
Comparison of band intensities of AFGP7 and 8 in the stomach fluids (lanes 1 and 7) for the two types of food shows that the food type has an influence on the concentration of the different AFGP sizes in these fluids. The stomach fluid of A. glacialis that had ingested B. saida contains higher concentrations of AFGP7 and 8 (Fig. 2, arrow c and d) than when A. glacialis had ingested crustaceans (Fig. 2, arrow e and f).
Furthermore, the gel electrophoresis, where all lanes were loaded in equal total concentration of AFGP, indicated that absorption or possible degradation of AFGP occur during the food passage through the digestive system. This is seen by the decrease in intensity of the AFGP7 and 8 bands from stomach to hind-gut fluid (Fig. 2, lanes 13 and 79), indicating the disappearance of AFGP7 and 8 from the fluids. The observation is supported by the increase of the intensities of the high molecular AFGPs (Fig. 2, lanes 13 and 79, arrow g).
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Discussion |
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During the dissection and sample preparation of the specimens of A.
glacialis it was noticed that about 50% of the specimens had ingested
Boreogadus saida, indicating that A. glacialis does not feed
exclusively on crustaceans as reported elsewhere
(Süfke et al., 1998).
This observation is supported by diet studies of A. glacialis and
large specimens of B. saida captured in Dove Bay, Northeast-Greenland
(K. Præbel, unpublished), which revealed that both species are
piscivorous and cannibalistic predators.
Our results show that there is a correlation between ingested food and the thermal hysteresis in the intestinal fluids of A. glacialis. Only non-significant osmolality differences of the gastrointestinal fluids were observed when the two food types were compared. Corresponding comparison of ion concentrations revealed that only stomach fluid differed significantly. This is probably due to the high ionic content in the crustaceans as they are iso-osmotic to seawater, and to the large amount of protons needed to buffer the carbonate originating from the cuticle of those animals.
One interesting question that arises from the results concerns the
extremely high antifreeze activity (2.04±0.31°C, HFP:
3.27±0.30°C) found in the mid-gut fluids where the ingested
food was B. saida. The physiological significance in terms of
freezing avoidance is doubtful, because the average freezing point of Arctic
seawater is 1.8°C (Garrison,
1998). The polyacrylamide gel electrophoresis showed that the
AFGPs are degraded into many sub-sizes during the digestion process. As
pointed out in other studies, the size composition of AFGP have crucial
influence on the antifreeze activity
(Ahlgren and DeVries, 1984
;
Kao et al., 1986
;
Osuga et al., 1978
;
Schrag et al., 1982
). Thus, a
possible explanation for the high antifreeze activity can be the combination
of many sub-sizes of AFGP and the fact that the concentration of AFGP
increases in the intestine due to water uptake by the gut wall
(O'Grady et al., 1982b
).
An increase in antifreeze activity was observed from the stomach to the mid-gut with a decrease in the hind-gut. This observation is consistent with the digestion, absorption and evacuation pattern. The antifreeze activity is low in the stomach due to little release of AFGP from the partly digested food. The increasing antifreeze activity in the mid-gut must be a consequence of increased release of AFGP from the digestion of the AFGP-laden polar cod, many sub-sizes of AFGP and increasing AFGP-concentration due to water uptake. Thus, the lower antifreeze activity found in the hind-gut might be caused by further degradation of the large sizes of AFGP and to the presence of less active smaller sizes. Nevertheless, the hysteresis freezing point of the hind-gut fluids is still well below of that of seawater.
From the gel electrophoretic study it is clear that AFGP from digested
B. saida were present in all the samples, also in the samples where
the food was believed to be crustaceans. Sæther et al.
(1999) showed that the total
gastrointestinal time for evacuation of an inert marker in B. saida
was approximately 400 h at 0.5°C. A similar evacuation time could be
expected for A. glacialis, since they live in colder environmental
temperatures and the digestion time decreases with temperature. Therefore, the
AFGP from B. saida found in all the samples confirms that A.
glacialis feed on other prey than crustaceans.
In view of the fact that fish are able to absorb intact proteins up to at
least 40 kDa (Berge et al.,
2003; McLean and Ash,
1987
; McLean et al.,
1999
), it is an open question whether or not absorption of
antifreeze glycoproteins from the intestinal fluids occurs in A.
glacialis. Experiments conducted by O'Grady et al.
(1983
) indicated that
antifreeze glycoproteins are not absorbed as intact molecules in the
intestinal system of the Antarctic notothenioids. Nevertheless, several
observations shown in the present study indicate that absorption of AFGP might
occur in A. glacialis. First, the gel electrophoretic study shows
decreasing intensity of the low molecular mass AFGP from stomach to hind-gut,
suggesting that absorption might be taking place. Secondly, our results show
that the antifreeze activity is lower in the hind-gut fluids compared with the
mid-gut fluid. Third, the high AFGP concentration combined with a long
evacuation rate will increase the possible AFGP absorption
(Hirst, 1993
). Thus, further
experiments on the relation between AFGP concentration, absorption and food
type are needed to answer the question of whether AFGPs are absorbed or
not.
In conclusion, the findings illustrate that the antifreeze activity in the intestinal tract of A. glacialis is dependent on food type. Furthermore, the results indicate that absorption of AFGP might occur in the digestive system of A. glacialis unlike that reported in the Antarctic notothenioids.
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Acknowledgments |
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References |
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