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First published online January 19, 2006
Journal of Experimental Biology 209, 407-420 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02008

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Freezing resistance of antifreeze-deficient larval Antarctic fish

Paul A. Cziko¹, Clive W. Evans², Chi-Hing C. Cheng^1,* and Arthur L. DeVries¹

¹ Department of Animal Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
² Molecular Genetics and Development, School of Biological Sciences, University of Auckland, Auckland, New Zealand

View larger version (41K): [in a new window]   Fig. 1. Geographic location (A) and schematic of the collection sites (B) of the fish eggs. P. antarcticum (Pa) eggs were collected from the platelet ice in Terra Nova Bay. G. acuticeps (Ga) eggs were found on the shallow bottom near McMurdo Station on Ross Island during several seasons. P. borchgrevinki (Pb) eggs were found in a crevice in the side of an iceberg near Cape Evans, Ross Island. All eggs were collected from below sea ice cover.

View larger version (11K): [in a new window]   Fig. 2. Cooling chamber used to determine the freezing resistance of G. acuticeps larvae. External ice was applied with a frozen toothpick; internal ice was introduced by touching the caudal peduncle with a cold needle. (A) Cross-section showing the placement of the inlet (IT) and outlet (OT) thermocouples, magnetic stirring bar (MS), false bottom (FB) and the cooling jacket (CJ) through which coolant from a refrigerated bath was circulated. (B) Top view. The larva is approximately 12 mm long.

View larger version (57K): [in a new window]   Fig. 3. Method used to determine the freezing resistance of P. antarcticum larvae. An individual larva was placed in a drop of seawater on a solid 6 mmx6 mm aluminum slide (AS), which was mounted on the thermoelectric cooling module (CS) of the Clifton nanoliter osmometer. A needle (ND) was cooled in liquid nitrogen and used to initiate ice crystal (IC) growth in the seawater surrounding the larva. Cooling is accomplished with stacked Peltier devices (PD) mounted on a water-cooled brass heat sink (HS). The stage temperature is controlled via a negative-feedback mechanism using a micro-thermistor (MT) mounted within the stage, and a separate temperature control module, connected to the cooling module by a cable (CC).

View larger version (22K): [in a new window]   Fig. 4. Seawater temperatures at McMurdo Station. Seawater temperatures were recorded from August 2002 to March 2004 at 40 m depth near a spawning site of G. acuticeps at the McMurdo Station. Arrows indicate approximate hatching and spawning dates for the yearly cohorts. G. acuticeps spawns in mid-November and embryonic development is protracted across c. 10 months. Hatching is in early September, near the start of the austral spring. The seawater is within 0.05°C of its freezing point (-1.91°C) between June and December, and shows little variation in temperature with depth during this period.

View larger version (25K): [in a new window]   Fig. 5. Developmental trends in the TH of body fluids of G. acuticeps larvae. (A) Serum MP (squares) and FP (circles) of larvae collected and reared in 2002 and 2003. (B) Intestinal fluid MP (triangles) and FP (squares) of the 2002 cohort only. Intestinal fluid MP is partially dependent on feeding status, which may account for the decrease in FP in the oldest larvae. (C) Serum TH did not increase significantly from 0 to 30 d.p.h. For larvae 30 d.p.h., TH was positively correlated with age, increasing by 0.008°C per day (linear regression, r2=0.77). (D) Intestinal fluid TH; TH did not change significantly throughout the intestinal fluid sampling period (84 d.p.h.). For G. acuticeps, the yolk-sac is completely absorbed by about 15 d.p.h. The broken line (A,B) indicates the seawater temperature at the time of hatch (-1.91°C). Values are means ± s.e.m. (serum samples, N=132; intestinal fluid samples, N=75).

View larger version (145K): [in a new window]   Fig. 6. Morphology of the gills of larval notothenioids. The gill arches of 1 d.p.h. P. antarcticum (A) and P. borchgrevinki (B) larvae were found to completely lack even rudimentary filaments. G. acuticeps larvae of the same age were found to possess developing gill filaments (C; arrows). Lamellae (arrowheads) form later in development, as illustrated by their presence in the gills of 70 d.p.h. G. acuticeps larvae (D). c, cartilaginous gill arch. Scale bars, 100 µm.

View larger version (45K): [in a new window]   Fig. 7. Phylogenetic analysis for confirmation of larval identities. An unrooted consensus tree resulting from neighbor-joining analysis of the complete 1047 nt mtND2 gene sequence from adults of several species within the family Nototheniidae (Notothenioidei), and the larvae collected at Terra Nova Bay and Cape Evans. The positions of the larvae within the tree identified them as Pleuragramma antarcticum and Pagothenia borchgrevinki (arrows). Adult individuals of the same species are indicated by brackets. Bootstrap values (1000 pseudoreplicates) are presented, but within-species values have been omitted for clarity. The scale bar indicates the relationship between branch length and the number of nucleotide differences between individual gene sequences.