Aquaculture 188 Ž2000. 91–101
www.elsevier.nlrlocateraqua-online
Freeze susceptibility in haddock žMelanogrammus
aeglefinus/
K. Vanya Ewart a,) , Brian Blanchard a , Stewart C. Johnson a , Wade
L. Bailey b, Deborah J. Martin-Robichaud c , Maria I. Buzeta c
a
c
NRC Institute for Marine Biosciences, 1411 Oxford St., Halifax, NS, Canada B3H 3Z1
b
KDF Consulting Ltd., St. John’s, NF, Canada
Department of Fisheries and Oceans Canada Biological Station, St. Andrews, NB, Canada E0G 2X0
Received 12 July 1999; received in revised form 4 December 1999; accepted 20 December 1999
Abstract
Haddock Ž Melanogrammus aeglefinus . is a promising new species for aquaculture in Eastern
Canada. Because haddock aquaculture could involve overwintering of fish in sea cages in icy
near-shore areas, haddock freeze resistance was studied. Measurements were done on the blood of
wild haddock collected during the winter from Georges and St. Pierre Banks as well as from
cultured haddock originating from Northeast Bank in the Bay of Fundy. Freezing points were
within the normal range for teleost blood, suggesting no freeze resistance adaptations. Glycerol in
blood samples from Georges Bank and from most cultured haddock was not above normal
physiological levels whereas St. Pierre Bank and Sandy Cove fish showed elevated glycerol
levels. However, glycerol in the latter group was far lower than amounts required to depress the
blood freezing point to a safe level in icy seawater. Thermal hysteresis measurements revealed no
antifreeze protein ŽAFP. activity in any of the samples. However, ice crystal morphology revealed
small amounts of AFP in a haddock sample collected in sub-zero seawater. The haddock
antifreeze differed from the antifreeze glycoproteins ŽAFGPs. of other gadids in that it was active
in the presence of peanut lectin and inactivated by EDTA. Neither the glycerol levels nor the trace
level of AFP shown here would be sufficient to protect this species from freezing in icy seawater
during winter. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Haddock; Melanogrammus aeglefinus; Antifreeze protein; Antifreeze glycoprotein; Thermal
hysteresis; Freezing point; Glycerol; Aquaculture
)
Corresponding author. Tel.: q1-902-426-7091; fax: q1-902-426-9413.
E-mail address: [email protected] ŽK.V. Ewart..
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 0 0 . 0 0 3 2 5 - 2
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K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
1. Introduction
Haddock Ž Melanogrammus aeglefinus . are found in cool north temperate waters at
temperatures ranging generally from 18C to 138C and depths normally below 27 m
ŽScott and Scott, 1988.. The natural habitat of haddock would present little or no
exposure to sub-zero seawater temperatures and ice crystals. Therefore, there would be
virtually no risk of freezing. However, the development of haddock aquaculture in sea
cages will require holding the fish in icy near-shore waters where fish can freeze.
Because of this risk, it is important to determine whether haddock are equipped to resist
freezing.
The risk of freezing in icy coastal waters arises from the different solute concentrations in fish and in seawater. According to colligative principles, solute molecules lower
the freezing point of a solution in a predictable fashion based on their concentration in
solution. This is a colligative property shared by all solutions. Teleost fish have
sufficient dissolved solutes in their fluids to lower the freezing point from about y0.68C
to y0.88C, whereas seawater, depending on the solute concentration, will freeze at
temperatures as low as y28C. Thus, when seawater reaches its freezing point, the fish
present in that water are actually below their freezing points ŽScholander et al., 1957.. In
the absence of ice, these fish would be unlikely to freeze because liquids can ‘‘supercool’’ Ži.e., to remain liquid, at temperatures below the thermodynamic freezing point. if
there are no ice nuclei on which ice can grow. However, if ice crystals are present in the
water, they can act as nuclei or templates for further rapid ice growth and fish can easily
freeze in this situation. In winter, the coastal waters of Atlantic Canada often present
these icy conditions under which fish can freeze and die. Consequently, the geographic
range of commercial aquaculture of freeze-susceptible species such as salmonids is
severely limited to small areas where the risk of freezing is minimal.
Temperate fish species present a variety of adaptations that confer freeze resistance.
Life history and behavioural adaptations include migration to deeper or warmer water
where freezing will not occur. Others, including antifreeze proteins ŽAFP. and glycerol
production, are biochemical adaptations that allow fish to remain in coastal waters and
avoid freezing ŽRaymond, 1993; Fletcher et al., 1998.. The AFPs lower the freezing
temperature of the fish non-colligatively by binding to ice crystals and inhibiting their
growth whereas glycerol acts as a colligative antifreeze agent. The AFPs are extremely
diverse among fish and five structurally different types are known to date ŽEwart et al.,
1999.. These include AFP types I–IV and antifreeze glycoproteins ŽAFGPs.. An early
study on the freezing points and antifreeze activities in marine fishes reported no
evidence of antifreeze in haddock ŽDuman and DeVries, 1975.. However, the study also
failed to detect antifreeze activity in cod Ž Gadus morhua.. More recent studies using
sensitive measurement techniques revealed that cod do produce abundant AFGP ŽGoddard and Fletcher, 1994.. Therefore, the possibility of antifreeze presence in haddock
should be re-investigated using the most sensitive techniques and glycerol accumulation
should also be examined.
The present study was conducted in order to determine whether haddock have
biochemical adaptations that would allow them to avoid freezing in sea cage culture on
the Atlantic coast of Canada.
K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
93
2. Materials and methods
2.1. Sample collection
Blood plasma samples were obtained from 10 adult haddock during winter Ž17–26
February 1998. on Georges Bank. Blood sera were also collected from two haddock on
4 December 1987 and six haddock on 16 December 1997 off Newfoundland in NAFO
division 3Ps ŽSt. Pierre Bank.. Water temperatures were not determined. These samples
were stored frozen until use.
Blood plasma samples were collected in early spring Ž27 March–22 April 1997. from
14 mature adult haddock at the Department of Fisheries and Oceans ŽDFO. St. Andrews
Biological Station in St. Andrews, NB. They were held at temperatures above 48C.
These fish had been obtained on the Northeast Bank in the Bay of Fundy ŽNAFO 5Y..
Sera were obtained from 10 juvenile haddock held in a sea cage at Fairhaven, NB, on 23
March 1998 at water temperatures from 18C to 38C and six juvenile haddock held at the
NRC Sandy Cove Aquaculture Research Station in NS on 24 February 1999 at a water
temperature of y0.58C. These juveniles were also progeny of the fish from the
Northeast Bank in the Bay of Fundy. Samples were stored frozen until use. Sera were
also obtained from three adult cod on Browns Bank on 6 April 1999 and stored frozen.
Sera and plasma have not been shown to differ with respect to antifreeze activity,
glycerol concentration, or freezing point and are equivalent for the purposes of this
study.
2.2. Glycerol measurement
Glycerol concentrations were measured using an enzymatic kit-based method as
described by Raymond Ž1992. and Driedzic et al. Ž1998.. Briefly, 500 ml of a glycerol
reagent solution ŽSigma Diagnostics 337-40A. was added to 5 ml of blood serum or
plasma, mixed, kept at room temperature for 5 min and the absorbance at 540 nm was
measured. Quantitation ŽmM. of glycerol employed a curve obtained from glycerol
standards. A pooled plasma sample from rainbow smelt Ž Osmerus mordax ., a species
known to accumulate glycerol to high Žprotective. levels during winter ŽRaymond,
1992., was measured for comparison.
2.3. QuantitatiÕe antifreeze actiÕity measurement
AFPs and AFGPs depress the freezing temperature while having a negligible effect
on the melting temperature in solution. The interval between these temperatures is
measurable and it is called a thermal hysteresis, or commonly, antifreeze activity.
Antifreeze activities were measured using a Clifton nanoliter osmometer ŽClifton
Technical Physics, Hartford, NY. as described by Kao et al. Ž1986.. For each data point,
triplicate measurements of freezing and melting temperatures were made by determining
temperatures of ice crystal growth Žfreezing. and shrinking Žmelting. viewed on a
cooling stage using a compound microscope ŽZeiss.. Blank hysteresis values, obtained
by measurements using water alone, were used to correct for the background error of the
94
K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
osmometer. Dilutions of pooled plasma from rainbow smelt, known to have an AFP
ŽEwart and Fletcher, 1990; Ewart et al., 1992., were used for comparison. Smelt plasma
was diluted in TCS ŽTris-buffered saline pH 7.4 containing 10 mM CaCl 2 ..
2.4. QualitatiÕe antifreeze actiÕity eÕaluation
Ice crystals grown in haddock blood samples were photographed using Polaroid film
on a microscope camera. Temperatures were maintained below the melting point to
ensure crystal stability or slow crystal growth. Care was taken to ensure that melting was
not taking place during photography because all crystals become circular during melting.
Crystal morphologies were evaluated as described by Hon et al. Ž1995. and Haymet et
al. Ž1999.. However, in this study, ice crystals in smelt plasma dilutions were used for
comparison.
2.5. AFP eÕaluation
Antifreeze type in blood samples was investigated using activity inhibition tests. The
activity of AFGPs is known to be mediated by their O-linked Gal–GalNAc dissacharides ŽYeh and Feeney, 1996.. Therefore, peanut lectin Ž0.5 mgrml., which binds
this carbohydrate, was added to serum to inhibit this activity. The activity of Ca2q-dependent type II AFPs requires divalent cations and can be inhibited by the addition of 30
mM EDTA ŽEwart et al., 1999.. If neither of the treatments inhibited antifreeze activity,
solubility in acetone and size fractionation were to be used to further determine whether
the antifreeze belongs to the other known fish types Žfish types I, III, or IV..
2.6. Statistical analysis
Blood freezing points, glycerol concentrations and thermal hysteresis data in all
groups were analysed using analysis of variance ŽANOVA.. When significant Ž p - 0.05.
differences were detected among groups using ANOVA, the Tukey–Kramer test was
applied to determine significant differences between specific groups.
3. Results
3.1. Glycerol leÕels in haddock blood
Glycerol levels in blood samples from mature haddock in St. Andrews, NB were
consistently below 0.1 mM, in line with normal physiological concentrations. Similar
levels of glycerol were present in blood from juvenile haddock in Fairhaven, NB and in
adult haddock obtained during the earlier sampling in division 3Ps on the St. Pierre
Bank off Newfoundland. However, significantly elevated glycerol levels were found in
haddock sampled in 3Ps 12 days later ŽTable 1.. The average glycerol concentration in
the latter samples was 5.3 mM. This is approximately 175 times higher than the trace
levels found in other haddock and it indicates a substantial glycerol elevation. Signifi-
Species, location and date
Haddock
St. Andrews Biological Station, DFO,U
27 March–22 April 1997
Fairhaven, NB,U 23 March 1998
Sandy Cove, NS,U 24 February 1999
St. Pierre Bank, 4 December 1997
St. Pierre Bank, 16 December 1997
Georges Bank, 17–26 February 1997
SmeltUU
Avalon Peninsula, NF, 20 February 1997
U
Freezing point Ž8C. Žmean"SD.
Glycerol ŽmM. Žmean"SD.
Thermal hysteresis Ž8C. Žmean"SD.
y0.710"0.058 a,b Ž ns14.
0.026"0.045a Ž ns14.
0.003"0.007 Ž ns14.
y0.633"0.064 a Ž ns6.
y0.688"0.073 a Ž ns6.
y1.074"0.088 c Ž ns 4.
y0.756"0.051b Ž ns6.
y0.577"0.047 d Ž ns10.
0.011"0.026 a Ž ns6.
3.082"1.400 b Ž ns6.
0.052"0.104 a Ž ns 4.
5.721"1.299 c Ž ns6.
1.100"1.345a Ž ns6.
0.001"0.003 Ž ns6.
0.004"0.003 Ž ns6.
0.004"0.004 Ž ns 4.
0.006"0.007 Ž ns6.
0.000"0.004 Ž ns10.
y1.208 Žpool.
177.2 Žpool.
0.333 Žpool.
K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
Table 1
Freezing points and thermal hysteresis determined by osmometry and glycerol concentrations determined by biochemical assay in the blood of haddock Ž M.
aeglefinus . collected from various sites.
There were no significant differences in thermal hysteresis detected by ANOVA among haddock groups. Glycerol and freezing point values from haddock within a
column with different superscript letters are significantly different Ž p- 0.05..
Originating from or progeny of Northeast Bank haddock.
Included for purposes of comparison.
UU
95
96
K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
cantly elevated glycerol levels Ž3.0 mM. were also found in haddock held at Sandy Cove
at a water temperature of y0.58C. However, even the concentration of 5.3 mM in the
3Ps haddock collected on 16 December would only contribute 0.018C to freezing point
depression, based on colligative properties. In contrast, a smelt plasma sample measured
in the same assay had 177 mM glycerol ŽTable 1. which would depress the freezing
point by 0.338C.
3.2. Antifreeze actiÕity in haddock blood
3.2.1. QuantitatiÕe antifreeze actiÕity measurement
There was no detectable thermal hysteresis in the haddock samples ŽTable 1..
Hysteresis values did not differ among groups ŽANOVA, p s 0.39. and they were
nearly identical to the blank value. These samples were measured directly without
dilution. For comparison, samples of smelt blood were measured without dilution or
following serial dilution in TCS. Undiluted smelt blood plasma showed substantial
hysteresis ŽTable 1.. The 0.1 = dilution showed reduced hysteresis and the 0.01 = and
0.001 = dilutions showed very low Žtrace. levels of hysteresis Žnot shown..
3.2.2. QualitatiÕe eÕaluation of antifreeze actiÕity
Antifreeze activity was not detected in most haddock blood samples by ice crystal
morphology studies ŽFig. 1.. However, one of the six haddock samples from Sandy
Fig. 1. Ice crystal morphologies in representative haddock blood samples from different locations. Photomicrographs were prepared as described in Materials and methods. Ice crystals in water, in TCS, and in smelt
plasma dilutions are shown as controls. The arrowhead indicates an ice crystal diagnostic of antifreeze activity
in a haddock sample from the Sandy Cove Aquaculture Research Station. Ice crystals in other haddock
samples from the Sandy Cove location did not show this specific morphology.
K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
97
Cove presented unequivocal evidence of antifreeze presence. Ice crystals in this serum
were faceted hexagons showing the six prism planes characteristic of crystals in
solutions of low concentrations of antifreeze ŽFig. 1.. In another sample from this group,
the ice crystals were slightly hexagonal but without crisp prism planes Žnot shown.. This
sample could be considered as equivocal. Smelt blood dilutions were evaluated in the
same way as the haddock blood. The hexagonal or bipyramidal ice crystals expressing
clearly defined planes diagnostic of antifreeze activity were evident in all dilutions of
smelt blood ŽFig. 1.. The haddock sample with clear antifreeze activity showed a crystal
similar to those in 1r100 and 1r1000 dilutions of smelt blood. Thus, antifreeze is
present at low concentrations in haddock exposed to sub-zero temperatures.
3.2.3. Examination of antifreeze type
The antifreeze type in the active haddock sample was studied using chemical
inhibition. Peanut lectin had not been previously used to inhibit cod antifreeze although
it is known to bind the appropriate dissacharide. Therefore, cod serum was used as a
positive control. In the presence of lectin, trace antifreeze activity in cod blood, as
evidenced by ice crystal morphology, was inhibited ŽFig. 2.. This is consistent with the
presence of the cod AFGP. In contrast, haddock antifreeze activity was not inhibited by
lectin, indicating that it is not an AFGP. As expected, EDTA had no effect on cod
antifreeze activity. However, it did inhibit the antifreeze activity in haddock.
3.3. Freezing point of haddock blood
The freezing points of blood plasma or serum samples were obtained in the process
of making quantitative antifreeze activity measurements. The freezing points of haddock
samples are shown in Table 1. Freezing points of the fish from the earlier sampling in
Newfoundland were depressed slightly. This was a colligative effect because no
hysteresis was detected. Glycerol levels did not account for the depression. These results
indicate a slight freezing point depression that is most likely the result of elevated
Fig. 2. Inhibition of antifreeze activity in blood samples for protein identification. EDTA Ž30 mM. and peanut
lectin Ž0.5 mgrml. were used as inhibitors. Cod and haddock sera are indicated.
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K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
electrolyte levels. It is not clear whether this reflects osmotic stress ŽSaunders et al.,
1975. or a physiological adaptive seasonal variation in electrolyte levels ŽFletcher,
1977..
4. Discussion
This study aimed to determine whether haddock are naturally equipped at the
biochemical level to resist freezing in icy seawater. Survival of teleost fish under these
conditions is favoured by the accumulation of antifreeze andror glycerol to levels that
depress the freezing point of the fish. In salmonids, whole animal lethal freezing points
are only slightly lower Ž0.04–0.208C. than the blood freezing points ŽFletcher et al.,
1988.. Therefore, blood freezing points are also a useful predictor of lethal freezing
limits in teleost fish. Haddock from different regions of the western North Atlantic,
including Georges Bank, the Northeast Bank ŽBay of Fundy., and the St. Pierre Bank,
were sampled in this study. The stock structure of haddock across these regions is
unclear. Mitochondrial DNA of haddock from some of these banks revealed no
significant differences in genotype frequencies in pairwise comparisons ŽZwanenburg et
al., 1992.. However, a geographic cline was detected in one genotype group and there
was also a marked trend towards increased genetic difference with geographic distance
ŽZwanenburg et al., 1992.. Thus, although haddock from different regions were considered separately in the freeze resistance analyses, it might be equally valid to combine
them.
Glycerol levels observed in haddock were far below those found in smelt and they
were not sufficient to cause relevant freezing point depression. However, levels varied
considerably among groups sampled. Glycerol was elevated in the blood of haddock
collected at Sandy Cove, NS on 24 February 1999 and in haddock from the St. Pierre
Bank on 16 December 1997, whereas fish collected from the St. Pierre Bank 12 days
earlier showed no glycerol accumulation. Glycerol levels in haddock samples were only
up to just above 5 mM and would only depress the freezing point by a few hundredths
of a degree. These concentrations are consistent with those obtained in another gadid,
saffron cod Ž Eleginus gracilis ., and in the Pacific herring Ž Clupea harengus pallasi .
ŽRaymond, 1992..
No evidence of quantifiable antifreeze activity Žthermal hysteresis. was found in
haddock. However, lower levels of antifreeze can be detected through ice crystal
morphology observations. Planar hexagonal faces are expressed on ice crystals in the
presence of antifreeze concentrations two to three orders of magnitude lower than those
at which full hysteresis is evident ŽKnight et al., 1984; Hon et al., 1995.. This effect was
illustrated in the ice crystal morphology of the smelt plasma sample and dilutions. One
haddock sample showed clear evidence of a low level of AFP or AFGP as evidenced by
ice crystal morphology and another sample from the same group showed equivocal
activity in that crystals were rounded hexagons. Ice crystals were round when grown in
samples from other haddock indicating no antifreeze activity.
The detection of antifreeze activity exclusively in juvenile haddock held at a sub-zero
temperature is consistent with variations in antifreeze occurrence observed in other fish
K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
99
species. The production of AFPs and AFGPs in several species appears to correlate with
exposure to freezing seawater conditions. In general, only species that risk freezing
produce antifreezes and they tend to do so at developmental stages and during seasons
that coincide with maximal freezing risk. Populations within a species can have different
antifreeze levels that correspond to extent of freezing exposure. For example, winter
flounder from the Bay of Fundy have lower antifreeze levels than do Newfoundland
flounder and this reflects their antifreeze gene copy numbers ŽHayes et al., 1991..
Antifreeze levels can also vary over the course of development. Juvenile stages of cod
Ž G. morhua. and herring Ž C. harengus harengus ., which overwinter in-shore, have
much higher antifreeze levels than the adults ŽChadwick et al., 1990; Goddard and
Fletcher, 1994.. Finally, there are seasonal variations in antifreeze levels in different fish
species that are responsive to photoperiod andror temperature ŽFletcher et al., 1998..
The very low level of antifreeze in a haddock appears consistent with the life history
of this species. The distribution of haddock suggests a deep-water habitat at cool but not
freezing temperatures ŽScott and Scott, 1988. implying a rather limited exposure to icy
seawater compared to cod ŽGoddard and Fletcher, 1994.. It has been also reported that,
relative to the numbers of cod and haddock around Newfoundland, a far greater
proportion of haddock die in the winter and spring in cold coastal waters ŽTempleman,
1965.. Taken together, these findings suggest that haddock may not have freeze
avoidance adaptations necessary for survival in icy coastal water and the species may
instead avoid freezing by virtue of its habitat choice. Other species residing in low-risk
habitats, such as sea raven Ž Hemitripterus americanus. and cunner ŽTautogolabrus
adspersus., have relatively low antifreeze levels that vary remarkably among individuals
and, more strikingly, from year to year ŽG.L. Fletcher, personal communication; Fletcher
et al., 1984; Valerio et al., 1990.. Haddock can now be counted among them.
The type of AFP present in haddock was shown to be distinct from the AFGPs found
in other gadids ŽAnanthanarayanan, 1989. by its activity in the presence of peanut lectin.
Moreover, the haddock antifreeze activity was inhibited by EDTA, suggesting that it
requires the presence of a divalent cation for activity. The only AFPs known to be
inhibited by EDTA are the type II AFPs of herring and smelt ŽEwart and Fletcher, 1993;
Ewart et al., 1992.. Thus, the AFP of haddock is not the AFGP found in other gadids but
is an unrelated protein with characteristics consistent with type II AFPs. The haddock
AFP may be type II but it is unlikely that all antifreeze types of fish are known and it is
equally possible that haddock antifreeze is a new EDTA-sensitive type that remains to
be identified.
The results presented here suggest that haddock are not freeze-resistant and that their
culture will have to be restricted to areas that have been found suitable for salmon and
other non-resistant species. The presence of an AFP in haddock indicates that the species
has the genetic capacity to resist freezing. However, the low level of antifreeze found
here is far from the concentrations required to generate freeze resistance. The haddock
with antifreeze activity were from a rather southerly population ŽBay of Fundy.. More
northerly populations may produce more AFP if genetic differences and environmental
factors affect its accumulation. A thorough comparison of antifreeze presence and levels
in haddock at different stages of development and from different populations may reveal
differences that lead us to the most appropriate broodstock for aquaculture. However,
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K.V. Ewart et al.r Aquaculture 188 (2000) 91–101
biotechnology to increase freeze resistance in haddock may prove to be the only
recourse in expanding culture of this species beyond the geographic limits of salmonid
culture.
Acknowledgements
We thank Cindy Doherty ŽConnors Bros. Limited., Ron Melanson and Donna Viger
ŽNRC IMB. and Bill MacEachern ŽDFO. for their kind assistance with collection of
haddock blood samples. Pooled smelt blood plasma was a generous gift from Sally
Goddard and Garth Fletcher of ArF Protein Canada. We thank Terje Larsen ŽUniversity
of Tromso. and Bill Driedzic ŽMemorial University of Newfoundland. for advice on
measuring glycerol. Helpful discussions with Marilyn Griffith ŽUniversity of Waterloo.
and with Marshall Greenwell and Dave O’Neil ŽNRC IMB. are gratefully acknowledged. We thank Santosh Lall ŽNRC IMB. for carefully reviewing the manuscript. This
project was funded by the CanadarNew Brunswick aquaculture development program
for non-traditional species, Connors Bros. Limited and the NRC Institute for Marine
Biosciences. This is publication number 42320 of the National Research Council ŽNRC..
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