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Cullins, Tammy L.
Antifreeze proteins in pelagic fishes from Marquerite Bay (western Antarctica)
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by Tammy L. Cullins.
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Thesis (M.S.)--University of South Florida, 2008.
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ABSTRACT: The Southern Ocean is home to two major types of fishes: endemics in the suborder Notothenioidii and representatives of oceanic fish families that are widely distributed in the midwater and benthic environments elsewhere (e.g. bathylagids, myctophids, liparids, and zoarcids) In most regions of the coastal Antarctic, e.g. the Ross Sea, there is a distinct separation in the pelagic communities at the shelf break between the oceanics (off-shelf) and the endemics (on-shelf). Coincidentally, in much of the coastal Antarctic, the shelf break also marks the boundary between a water column entirely composed of the very cold (-2Â¨C ) Ice Shelf Water and an oceanic profile that includes warmer Circumpolar Deep Water (2Â¨C at 200 m) at intermediate depths. The distinct separation in pelagic communities observed in most coastal regions of the Antarctic is not seen on the western Antarctic Peninsula (WAP), where circumpolar deep water intrudes to form a warmer midwater and oceanic species are strongly represented. It was hypothesized that the cold ice-shelf water, lethal to fishes without antifreeze glycoproteins (AFGP's) in their blood, was excluding the oceanic species from most of the Antarctic continental shelf waters. To test the hypothesis, nine species of fish captured in WAP shelf waters were tested for the presence of AFGP's. The oceanic fish families analyzed: Myctophidae (Electrona and Gymnoscopelus), Zoarcidae (Melanostigma), Gempylidae (Paradiplospinus), Paralepididae (Notolepsis), and Bathylagidae (Bathylagus) showed no antifreeze activity. Two endemic species captured in the same sampling program did show antifreeze activity: the important pelagic species Pleuragramma antarcticum (Nototheniidae) and the Bathydraconid (Vomeridens). The absence of AFGP's in the blood of Antarctic oceanic species makes a strong case for temperature exclusion of oceanic fishes in the coastal Antarctic.
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Advisor: Joseph J. Torres, Ph. D.
x Marine Science
t USF Electronic Theses and Dissertations.
Antifreeze Proteins in Pelagic Fishes from Marquerite Bay (Western Antarctica) by Tammy L. Cullins A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major Professor: Joseph J Torres, Ph.D. David Mann, Ph.D. Arthur DeVries, Ph.D. Date of Approval: June 23, 2008 Keywords: Antifreeze peptides, Pleuragramma antarcticum nanoliter osmometer, midwater fish, GLOBEC Copyright 2008, Tammy L. Cullins
i Table of Contents List of Tables ii List of Figures iii Abstract iv Introduction 1 Methods and Materials 5 Results 9 Discussion and Conclusions 12 References 17
ii List of Tables Table 1: Mean melting point, freezin g point and thermal hysteresis of fish sampled 9
iii List of Figures Figure 1: GLOBEC III cruise track and study area 6 Figure 2: ANOVA 10 Figure 3: Duncan Multiple Range Test Homogenous Groups 10
iv Antifreeze Proteins in Pelagic Fishes from Marquerite Bay (Western Antarctica) Tammy L. Cullins ABSTRACT The Southern Ocean is home to two major types of fishes: endemics in the suborder Notothenioidii and representatives of oceanic fish families that are widely distributed in the midwater and benthic environments elsewhere (e.g. bathylagids, myctophids, liparids, and zoarcids) In most regions of the coastal Antarctic, e.g. the Ross Sea, there is a distinct separation in the pelagic communities at the shelf break between the oceanics (off-shelf) and the endemics (on-shelf). Coincidentally, in much of the coastal Antarctic, the sh elf break also marks the bounda ry between a water column entirely composed of the very cold (-2C ) Ice Shelf Water and an oceanic profile that includes warmer Circumpolar Deep Water (2C at 200 m) at intermediate depths. The distinct separation in pelagic communities observed in most coastal regions of the Antarctic is not seen on the western Antarctic Peninsula (W AP), where circumpolar deep water intrudes to form a wa rmer midwater and oceanic sp ecies are strongly represented. It was hypothesized that the co ld ice-shelf water, lethal to fishes without antifreeze glycoproteins (AFGPs) in their blood, was ex cluding the oceanic sp ecies from most of the Antarctic continental shelf waters. To test the hypothesis, nine species of fish captured in WAP shelf waters were tested for the presence of AFGPs. The oceanic fish
v families analyzed: Myctophidae ( Electrona and Gymnoscopelus), Zoarcidae ( Melanostigma ), Gempylidae ( Paradiplospinus ), Paralepididae ( Notolepsis ), and Bathylagidae (Bathylagus) showed no antifreeze activity. Two endemic species captured in the same sampling program did show antifre eze activity: the important pelagic species Pleuragramma antarcticum (Nototheniidae) and the Bathydraconid ( Vomeridens ). The absence of AFGPs in the blood of Antarctic oceanic species makes a strong case for temperature exclusion of oceanic fi shes in the coastal Antarctic.
1 Introduction The Southern Ocean is home to two major types of fishes: endemics in the suborder Notothenioidii and representatives of oceanic fish families that are globally distributed in the midwater and benthos (e.g. bathylagids, myctophids, liparids, and zoarcids (Andriashev 1965, DeWitt 1965, DeWitt 1968, DeWitt 1970, DeWitt 1971, Ekau 1990, Hubold 1991, Clarke & Johnst on 1996, Greely et al. 1999). The notothenioidii are primarily benthic as a dults with the exception of the Antarctic silverfish, Pleuragramma antarcticum and the toothfish, Dissostichus mawsoni which are both found in the coastal pelagic zone (Andriashev 1965, DeWitt 1965, DeWitt 1968, Andriashev 1970, DeWitt 1971, Hubold 1991, Ea stman 1993, Clarke & Johnston 1996, Eastman 1997,). The coastal benthos is domin ated by the notothenio ids with a few other families (e.g. Rajidae, Muraenolepidae, Zoarcidae, Liparidae, and Bothidae) represented (Andriashev 1965, DeWitt 1965, DeWitt 1968, Andriashev 1970, DeWitt 1971, Hubold 1991, Eastman 1993, Clarke & Johnston 1996, Eastman 1997,). The shelf waters of the Western Antarctic Peninsula (WAP) have a very different faunal mix. Here there is a strong repr esentation by the Myctophidae, a globally distributed oceanic family (Donnelly & Torres 2008). This difference in faunal
2 representation may be a result of the warmer profile found in the water column. Because of its position relative to the Antarctic Ci rcumpolar Current, wa rm Circumpolar Deep Water (CDW) (2C) episodically intrudes onto the shelf, making the intermediate depths (200-400 m) warmer here than in the remainde r of the coastal Antarctic (Dinniman and Klinck 2004, Donnelly & Torres 2008). For example, the shelf regions of the Ross and Weddell Seas are both uniform in temperature (-2C) from top to bottom because of the very cold ice-shelf water that dominates the water column in both locations (Dinniman et al. 2003, Donnelly et. al 2003). The uniformly cold water likely has a profound influence on the faunal diversity in most of the coastal Antarctic. Fishes in the perciform suborder Notothen ioidii are able to su rvive in the cold shelf waters of the Ross and Weddell Seas b ecause of a unique adaptation: the presence of biological antifreezes in their blood, w ithout which they would freeze (Devries and Wohlschlag 1969, Devries 1970, Devries & Lin 1977, Devries 1986, Devries & Cheng 2005, Cziko et al. 2006,). Like most vertebrates, fishes have an internal osmotic pressure (OP) of 300-500 mOsm, about 1/3 to that of seawater (Devries and Lin 1977, Hickman and Trump 1969). Because of their low OP, w ithout the aid of the antifreeze compounds their body fluids should freeze long before th e seawater surrounding them. The antifreeze which the notothenioids possess prevents ice crystals from propagating in their blood and other body fluids (Devries and W ohlschlag 1969, Devries 1970, Devries & Lin 1977, Devries 1986), thereby allowing them to survive in the ice-laden waters that would be rapidly lethal to most fishes.
3 Antifreezes are fairly sma ll molecules (2,600-33,700 daltons), that can be either peptides or glycoproteins. Antifreeze protei ns (AFP) were the first to be discovered (Devries and Wohlschlag 1969). There are at least 8 different size classes of glycoproteins with 1 being the largest ( 33,700 daltons) and 8 being the smallest (2,600 daltons). All of the AFGPs consist of an alanine/proline-alanine-threonine backbone with threonine O-glycosylated by a dis accharide (Devries and Cheng 2005); The two smallest size classes are normally the most abundant in the body fluids. The same antifreeze glycoproteins are found not only in the notothenioids, but also in several northern Arctic gadids. Antifreeze peptides (A FP) were first identified in the winter flounder, Pseudopleuronectes americanus and can be found in Artic, northern temperate fish, and two Antarctic zoarcid fishes (Dev ries and Cheng 2005). Three more types of antifreeze peptides have been found since their initial discovery. These antifreeze peptides differ in protein sequence and secondary and tertiary structures, where as the AFGP only differ in the number of Ala/Pr o-Ala-Thr repeats (Devries and Cheng 2005). Both types of antifreeze (AFGP and A FP) work by binding to the face of a growing ice crystal and inhibi ting its growth (Devries 1986). When the antifreeze binds to the small seed crystal of ice, no growth is observed until the non-equilibrium freezing point or the thermal hysteresis freezing point is reached. At that point growth occurs as a rapid burst of spicules from the ice crystal. Th e spicules grow in the non-preferred axis of growth or c-axis. The concentr ation of AFGP also affects th e shape of ice crystal growth, with samples containing more AFGP having sm aller spicules and those with lower AFGP
4 concentrations having more of a starburs t/firework pattern of growth (personal observation). The antifreezes of the Antarctic notothenioids allow them to dwell in shelf waters throughout the Antarctic, where they ar e the overwhelmingly dominant group (Andriashev 1965, DeWitt 1970). The fact that oceanic fishes like the myctophids, paralepidids, and bathylagids are not found in most of the Antarctic coastal system, but can be found in the warmer waters of the WA P shelf, suggests that temperature may be playing an important role in their di stribution. (Andriashev 1965, DeWitt 1965, DeWitt 1968, DeWitt 1970, DeWitt 1971, Ekau 1990, Hubold 1991, Eastman 1993, Clarke & Johnston 1996, Eastman 1997, Greely et al 1999, Donnelly et. al 2003, Donnelly & Torres 2008). To investigate this possibility we examined 9 Antarctic fish species, including 5 oceanic species to determine if antifreeze was present in their blood.
5 Methods and Materials Fishes for the study were captured during th e fall process cruise of the Southern Ocean Global Ocean Ecosystems Dynamics Program conducted in the vicinity of Marguerite Bay (Fig 1), (SO GLOBEC III April 2002May 2002, ARSV Lawrence M. Gould) Antarctic Peninsula (Hofmann et al. 2004 ). Fishes were collected using either a 10m2 MOCNESS net system or a 2.25 m2 Tucker trawl, both w ith closed codends, to minimize damage to the specimens. Trawls were conducted from the surface to 1000m. Immediately upon recovery the cod ends were emptied and fish were extracted and placed on ice. Blood was collected by wicking it up into a heparinized capillary tube from the caudal vein. The blood was then frozen at -80C and shipped to St. Petersburg, FL where it was stored in a -80C freezer until an alysis. Samples were taken from the caudal veins of Pleuragramma antarcticum (N=20) Electrona antarctica (N=10) Gymnoscopelus nicholsi (N=5) G. braueri (N=5) Melanostigma gelatinosum (N=2) Paradiplospinus gracilis (N=1) Notolepis coatsi (N=1) Vomeridens infuscipinis (N=1) and Bathylagus antarcticus (N=1). All of the fish that had blood removed, as well as representatives of th e species caught, were also fro zen whole and shipped to Saint Petersburg, FL. All of the fishes analyzed were adults. Additional blood samples were
wicked out of the pericardial cavit y of the frozen fish analyzed Melanostigma gelatinosum (N=1), Vomeridens infuscipinis (N=1), and Bathylagus antarcticus (N=1). Figure 1: GLOBEC III crui se track and study area a) b) a) Global view of cruise track (red line) for GLOBEC III cruise from Chile to Western Antarctic Peninsula. 6 b) Study area cruise track (red line) show ing Antarctic circle (neon blue line).
7 Blood samples were analyzed using either a Clifton nanoliter osmometer or an Otago nanoliter osmometer. Before analysis each sample was thawed and approximately 5l of blood was drawn up into each of two 10 l capillary tubes. One end of each tube was sealed using a Bunsen burner and both tubes were spun down at 9000 RPMs in an Eppendorf micro-centrifuge. The Pleuragramma antarcticum samples were spun down for 1 minute and all other samples were spun down for 2 minutes be cause of the large concentration of tissue in the blood samples. Both tubes were removed and the bottom portion, which contains red blood cells and other material, was discarded. A sample was then drawn up into the sample loader: a hand drawn glass needle filled with Cargille Type A immersion oil. A small drop of sample was then placed into the center of all 6 wells in the sample holder, which had been f illed with Cargille Type B immersion oil and was located in the peltier apparatus that allo wed regulation of sample temperature. Each drop was approximately half the diameter of the well and as close to the center as possible. A small amount of dry nitrogen wa s blown across the sample holder to prevent condensation in the sample area. The sample compartment was covered with a glass cover slip and frozen solid using the machin es freezing cycle. A microscope was used to view the sample wells at 70x and a melting poi nt and freezing point were determined for each sample. The freezing cycle of the Otago nanoliter osmometer lowered the samples temperature to -20C. In order to determin e the melting point of a single sample, the temperature was manually raised to -2C. Fr om this point the temperature was slowly
8 raised until a single ice crysta l, spanning approximately of the diameter of the blood drop, remained and was stable. This point was recorded as the melting point for that sample. From here the temperature was lo wered approximately .01 C every 15 seconds until the ice crystal was seen to grow uncont rollably. This point was recorded as the freezing point of the sample. The difference (if any) between the melting point and freezing point was recorded as the thermal hys teresis. The six samples were again put through the machines freezing cycle and a diffe rent well chosen for analysis. This was repeated until all of the wells were analy zed. An average melting point, freezing point, and thermal hysteresis for that fish was then calculated as well as an overall average melting point, freezing point and thermal hysteresi s for all the fish analyzed from that species. The average thermal hysteresis for each fish analyzed was then entered into STATISTICA and an ANOVA was preformed. A Duncan Multiple Range Test was then preformed to determine if groups were homologous.
9 Results Table 1: Mean melting point, freezing point and thermal hysteresis of fish sampled Fish family Fish species N n SL (mm) MP (-C)aFP (C)a Hysteresis Osmotic Pressure of blood (mOms) b Nototheniidae Pleuragramma antarcticum 20 3-4 157 0.71 0.05 1.16 0.07 0.45 0.05 382 Myctophidae Gymnoscopelus nicholsi 5 2 155 0.75 0.08 0.82 0.08 0.07 0.01 404 Gymnoscopelus braueri 5 2 133 0.84 0.10 0.90 0.10 0.06 0.01 452 Electrona antarctica 10 2 94 0.77 0.08 0.84 0.08 0.06 0.01 414 Zoarcidae Melanostigma gelatinosum 2 3 160 0.56 0.01 0.63 0.01 0.07 0.01 301 Melanostigma gelatinosum (Whole fish) 1 4 172 1.04 0.01 1.14 0.07 .10 0.02 560 Gempylidae Paradiplospinus gracilis 1 2 360 0.88 0.02 0.95 0.01 0.07 0.01 474 Paralepididae Notolepis coatsi 1 3 178 0.89 0.15 0.96 0.15 0.07 0.02 479 Bathydraconidae Vomeridens infuscipinis 1 3 156 0.66 0.13 1.29 0.25 0.62 0.12 355 Vomeridens infuscipinis (Whole fish) 1 6 156 0.98 0.26 1.64 0.18 0.80 0.17 527 Bathylagidae Bathylagus antarcticus 1 2 158 0.84 0.01 0.91 0.01 0.07 0.02 452 Bathylagus antarcticus (Whole fish) 1 1 142 0.79 0.85 0.06 425
N = # of fish sampled, n = # of trials run per fish, SL is the average standard length of fish sampled for that species; a: mean 95% confidence interval; b: Osmotic pressure was calculated from the melting point readings. Figure 2 : ANOVA Figure 3: Duncan Multiple Range Test Homogenous Groups Species Hysteresis 1 2 2 Gymnoscopelus braueri 0.055660 **** 4 Electrona antarctica 0.063500 **** 5 Melanostigma gelatinosum 0.068315 **** 3 Gymnoscopelus nicholsi 0.073000 **** 1 Pleuragramma 0.488292 **** 10
11 The ice growth patterns seen in the sample s correlated with the level of hysteresis they exhibited. As soon as the solidly frozen samples began to melt, a difference was seen between the ice structure of Pleuragramma and Vomeridens when compared to that of the other fish species. Many straight lines could be observed in the ice crystal of Pleuragramma and Vomeridens and when the sample had melted down to a single stable crystal it appeared square, rectangular, or tr apezoidal in shape with clear facets present. In comparison, samples from the other fish c ontained almost no faceting. The solid piece of ice within the well broke into many sma ll spheres while it was melting with the final ice crystal being spherical or oblong in shape. Differences were also s een in the growth of the crystals. Ice growth for the Pleuragramma samples was from the edges (a-axis) of the seed crystal disc, which rese mbled a starburst pattern, with many spikes emanating from the central crystal in all directions. The ice growth pattern for Vomeridens infuscipinis was also planar in nature, but the pattern exhibited was hexagonal bi pyramidal, with the ice crystal growing from either end (caxis growth) to form a long spike. The ice growth pattern for all other species was spherical in nature with the ice crystals growing equally from all sides of the seed disc. The samples run from the frozen fish exhibited a higher osmotic pressure, but no other difference was seen.
12 Discussion and Conclusions The oceanic fish families analyzed: Myctophidae ( Electrona and Gymnoscopelus), Zoarcidae (Melanostigma ), Gempylidae (Paradiplospinus ), Paralepididae (Notolepis ), and Bathylagidae ( Bathylagus) showed no antifreeze activity. The small hysteresis that occurred was likely a result of lag between the readout and the actual temperature at the sample holder. A th ermal hysteresis of the same magnitude was also seen during some of the calibrations th at were preformed using deionized water, which should exhibit no hysteresis at all. Even if this hysteresis was not also seen in the deionized water the amount of antifreeze that would cause a hysteresis this small would offer no protection to the fish in such an ice laden environment as Antarctica. The Nototheniidae ( Pleuragramma ) and Bathydraconid (Vomeridens ), two species from predominantly Antarctic families, did show antifreeze activity. The ANOVA preformed clearly shows that there is a significant diffe rence between the nominal hysteresis seen in the oceanic and that seen in the endemics (Figure 2). This can also be seen in the Duncan Multiple Range Test where the oceanics grouped together and the Pleuragramma was excluded (Figure 3). The blood from the frozen Vomeridens was extracted to use as a standard to validate that the analysis procedure would work for blood drawn from frozen fish. The only difference seen was an increa se in osmotic pressure, which may result
13 from cell lysis due to freezing, as additional osmotic solutes were released from cells which have stores of solutes that do not contribute to the osmotic activity of the cell. Once the procedure was validated other spec ies of an appropriate length (>150mm SL) were analyzed. The lack of antifreeze activity in the oceanic familie s means that they will freeze and die when encountering ice crystals because of their supercooled state at the temperatures typical of the Ross and Wedde ll Sea shelves. However, on the WAP shelf where temperatures are typically above 0C in the intermediate depths (200-400 m) the oceanic fishes, and other fishes lacking antifreeze, are better able to survive. The low levels of antifreeze in Pleuragramma most likely only protect them from limited contact with ice which may be why th e majority of the silverfish population is found below 100 meters of depth (Lancraft et al 2004). The hysteresis observed in Marquerite Bay fish is lower (ave = 0.45) than that found in fish captured from McMurdo Sound (ave = 0.91) (Cziko et al. 2006) which could be a result of warmer temperatures in the WAP. Jin and Devries (2006) found that some Antarctic fish are ab le to adjust their AFGP levels based on temperature exposure. With more CDW ente ring Marguerite Bay the Pleuragramma may not need as much AFGP for protection because of the warmer layer (2C) which does not exist in McMurdo Sound. It is hard to compare the difference in hysteresis levels between this study and those conducted by Wohrmann (1995, 1996) because of the differences in tec hniques between the two studies.
14 AFGP presence was evident not only with the thermal hysteresis, but also in the nature of ice crystal growth and melting. Samples which contained AFGP ( Pleuragramma and Vomeridens ) showed a distinct ice-grow th pattern. When ice grew from a single crystal during the freezing point determination, growth was very fast once the thermal threshold was reached. The pattern of growth was also unique. In the case of Pleuragramma the ice would grow in a star-burst pa ttern with spikes emanating from the central crystal at multiple angles. The gr owth of ice crystals in the blood of Vomeridens was similar, but in the form of a hexagonal bi pyramide. It has been shown that this type of growth correlates with the presence of AFGP (Devries 1986) because the binding of the AFGP to the ice matrix constrains growth to a certain plane. Samples which did not contain AFPs ( Electrona antarctica, Gymnoscopelus nicholsi, G. braueri, Melanostigma gelatinosum, Paradiplospinus gracilis, Notolepis coatsi, and Bathylagus antarcticus ) melted down into spherical discs and ice grow th occurred equally along all edges of the disc. Based on the insignificant level of hysteresis seen in the midwater fish families ( Electrona antarctica, Gymnoscopelus nicholsi, G. braueri, Melanostigma gelatinosum, Paradiplospinus gracil is, Notolepis coatsi, and Bathylagus antarcticus ) and their pattern of ice crystal growth it can be concluded th at they do not contain AFPs to protect them from freezing in the ice laden Antarctic wate rs. The presence of oceanic species on the Western Antarctic Peninsula shelf but not on the Ross Sea or Weddell Sea shelves suggests that temperature is indeed the limiting factor in the distribut ion of oceanic fishes
15 in Antarctic waters. There is an abrupt transition from oceanic fauna to Antarctic endemics at the shelf break in the Ross Sea (Donnelly et al 2003, DeWitt 1970), the best studied of the Antarctic coastal regions with respect to fish distributions. Other than differences in the water column temperature there are few obvious differences that would stop oceanic species from residing in the shelf waters of the Ross Sea. For example, the depth of the shelf in the Ross Sea and Marquerite Bay are similar enough that depth would not be limiting. In 2003, Dinniman et al modeled the circulation in the Ross Sea and found that warm CDW does in fact in trude on to the shelf of the Ross Sea periodically which could in fact introduce oceanic species onto the Ross Sea shelf as on the WAP. However, the fishes mobility would allow access to the shelves of either region without the CDWs intrusion, whereas the benthic fishes are unlikely to because of their lack of mobility. One other item of interest to consider is the possible competition between Electrona antarctica and Pleuragramma antarcticum, the two dominant fish in the oceanic realm and coastal ecosystems respectively. Both fish have similar diets: copepods in their early life history and krill later in life (Hubold 1985, Lancraft et al. 2004), and they are both vertical migrator s with pelagic eggs. The differe nces between them occur in their growth and tim e of reproduction. E. antarctica lives for 4 years and reproduces in its last year (Greely et al 1999). It is long lived and slow growing in comparison to other myctophids, but not when compared to the nototheniid P. antarcticum Pleuragramma lives for 21 years and reproduces in its ninth year of life (Hubold and Tomo 1989).
16 Without the presence of antifreeze in P. antarcticum E. antarctica might be able to outcompete it in the coastal regions of the WAP. In fact, this competition may already be occurring because P. antarcticum is disappearing from the mid-regions of the Antarctic Peninsula as the peninsula warms and the ice cover is reduced. With the continued warming trend (Smith and Stammerjohn 2001, Ducklow et al. 2007), P. antarcticum may have a hard time competing in this region of Antarctica.
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22 Smith R, Stammerjohn S (2001) Variation of su rface air temperature and sea ice extent in the western Antarctic Peninsula regi on. Annals of Glaciology 33: 493-500 Wohrmann A (1995) Antifreeze glycopeptides of the high-Antarc tic silverfish Pleuragramma antarcticum (Nototheniodei). Comparative Biochemistry and Physiology 111C:121-129 Wohrmann A (1996) Antifreeze glycopeptides an d peptides in Antarctic fish species from the Weddell Sea and the Lazarev Sea. Marine Ecology Progress Series 130:47-59 Wohrmann A (1998) Antifreeze glycopeptides an d peptides in the freezing avoidance of Weddell Sea fishes: its relation to mode of life, depth distribution and evolution. In: Portner H, Playle R (eds) Cold Ocean Physiology. University Press, Cambridge Wohrmann A, Haselbeck A (1997) Adapta tions of the Antarctic silverfish Pleuragramma antarcticum to pelagic life in high-Antarc tic waters. Marine Ecology Progress Series 151:205-218