S .J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
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2. Materials and methods
2.1. Collection and maintenance of krill On 2 April 1999, approximately 500 Antarctic krill were collected from the marginal
ice edge near Casey Station, Antarctica 648170S, 1058380E, using a rectangular
2
mid-water trawl net RMT-8, mouth area 8 m . After the catch was sorted, viable krill were maintained in tanks aboard RSV Aurora Australis during passage, and on 21 April
were transferred to holding tanks at the Australian Antarctic Division, Kingston, Tasmania.
On 10 May, 48 krill were removed from the holding tanks and separated into three groups of 16 animals, each in a separate tank. These groups were designated the
‘starved’ group, which received no food throughout the experiment, the ‘PAR-only’ group, which received algae grown under PAR wavelengths, and the ‘PAR 1 UVA B’
group, which received algae grown under a full range of PAR, UVA and UVB wavelengths.
Each group was placed in separate flow-through tanks connected to a common seawater supply to ensure identical water quality. Within each tank, individual krill were
isolated in separate 2-l plastic jars that were perforated to allow water exchange. Water temperature was maintained at 0.58C 60.28C and 33 61 ppt salinity. Flow rate
21
through all tanks was approximately 2 l min .
2.2. Growth of algae Sixteen polystyrene culture flasks 600 ml were each filled with 450 ml of GP5
media and inoculated at 4–8 day intervals see Results, Table 2 with 150 ml of a high density, stationary phase culture of Phaeocystis antarctica obtained from Prydz Bay,
Antarctica, 768E, 698S. The culture remained in the colonial stage of the Phaeocystis life cycle for the entire experiment Marchant et al., 1991. The culture flasks were grown
under one of two light treatments: PAR-only, and a combination of PAR 1 UVA B see Results, Table 1 in a 12:12 h light:dark photoperiod.
A two-chambered Ratek growth cabinet maintained at 28C was used to provide
Table 1 Phaeocystis antarctica. Irradiation conditions for culture of dietary algae during a 12:12 h light:dark
photoperiod
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Treatment Wavelength range nm
Irradiance W m
22
UV 280–320 UVB
6.76310 320–400 UVA
4.96 400–700 PAR
11.81 PAR
280–320 UVB –
320–400 UVA –
400–700 PAR 11.23
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.J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
irradiance for algal growth. The upper chamber was fitted with two FSL 140 17 W UVB-emitting fluorescent tubes Philips
E, three ‘black light’ 17 W UVA tubes NEC, and four 15 W ‘cool white’ tubes Osram
E. The lower chamber was fitted with five 15 W ‘cool white’ tubes Osram
E. UVB tubes were covered in GrafixE cellulose acetate sheet to remove UVC , 280 nm wavelengths.
Light levels were monitored using a Solar Light E PMA 2100 radiometer with
quantum PAR, UVA, and UVB sensors. Levels were measured beneath a section of plastic cut from the flat surface of a culture flask. Cultures were irradiated at a distance
of approximately 40 cm from the light sources and flasks were inverted daily to prevent algal precipitation.
Because of inaccuracies encountered in counting cells of the colonial form of P .
antarctica, we adapted the method used by Riegger and Robinson 1997, who, in the absence of reliable data for cell numbers, normalised maximum absorbance in the UV
280–400 nm to maximum absorbance by chlorophyll a. We analysed methanol extracts of culture samples method adapted from Dunlap et al., 1995 to determine near-
instantaneous differences in the culture content of chlorophyll a and of UV-absorbing MAAs, without the time delay inherent in HPLC. In using this method we assumed that
the amount of chlorophyll a per cell remained constant throughout the experiment.
On the day of feeding, 40 ml of each culture was centrifuged for 20 min at 400 g, and the supernatant medium was discarded. The residual pellet of algae was extracted for 1 h
with 3 ml of 100 methanol, suspended insoluble material was removed by centrifuga- tion and UV visible spectra of krill extracts were recorded with a GBC scanning
spectrophotometer. All of the extracts showed a distinct absorption in the region 330–340 nm corresponding to the combined suite of algal MAAs. Chlorophyll
concentrations were estimated using the peak absorbance value at 663 nm refer to Results, Fig. 1.
In all cases, the chlorophyll a content was greater in cultures having the PAR treatment than for the UVB treatment. Therefore, the volume of feed given to each krill
in the PAR group was reduced by the fraction of the chlorophyll a absorbance in the UVB treatment compared with the chlorophyll a absorbance in the PAR treatment.
2.3. Feeding experiments On the day of feeding, and following extraction of algae to establish chlorophyll
MAA ratios, remaining PAR 1 UVA B culture from two 600 ml flasks of P . antarctica
culture inoculated 2 weeks previously was split evenly between krill in the UVB group, and a corresponding standardised volume of the PAR-only culture was fed to the PAR
group krill see Section 2.2. The control group krill received no food for the duration of the experiment. The volume of algae fed to the krill varied depending on the growth
state of the algae and the number of krill remaining. Krill were fed on a total of eight occasions for details and dates, refer to Results, Table 2. Feeding ceased after day 58,
whereupon all remaining krill were starved until analysis on day 98.
2.4. Extraction of MAAs from krill Animals were removed from the experimental enclosures on days 36, 63 and 98.
S .J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
97
Fig. 1. Phaeocystis antarctica. Spectrophotometric analysis of methanol extracts of P . antarctica grown under
PAR-only and PAR1UVA B irradiation standardised to chlorophyll a absorption at 663 nm.
Since any attempt to remove gut contents from the krill manually would have led to the removal of the entire digestive gland, we chose instead to allow a period prior to
analysis during which most material in the gut would be excreted. Given that the mean gut residence times of Isochrysis and Thalassiosira are 47 and 256 min, respectively
Pond et al., 1995, we assumed that 4 days of starvation 5760 min was sufficient to
Table 2 Phaeocystis antarctica. Volume of culture fed to each individual krill. Difference in culture volume is due to
standardisation to chlorophyll a absorbance maximum refer to Materials and methods section for details Feed
Date Day
Volume of culture 1999
fed to each krill ml UV
PAR 1
24 May 14
35 27
2 28 May
17 35
27 3
4 June 24
35 21
4 10 June
30 35
17 5
17 June 37
56 46
6 24 June
44 56
50 7
30 June 50
56 45
8 8 July
58 56
33
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.J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
allow most ingested material to leave the digestive tract, and we timed feeding and analysis to coincide with this period. Upon removal from the tanks, krill were
immediately weighed, segmented into six pieces of approximately 0.05–0.2 g and placed into a 15 ml extraction tube. All krill had a wet mass between 0.32 and 0.53 g.
Krill tissues were extracted by three serial extractions each using 2 ml of 100 methanol for 1 h Dunlap and Chalker, 1986. Following each extraction the contents
were centrifuged at 3000 g for 15 min and the supernatant decanted and pooled. The pooled extracts were passed through a C
Sep-Pak E cartridge Alltech to remove
18
intractable lipids and absorption spectra were obtained with a GBC scanning spec- trophotometer to determine the maximum optical density in the UV spectrum 280–400
nm. Three aliquots of 1 ml were transferred into 1.5 ml Eppendorf tubes and methanol was reduced on a Heto
E mini DNA vacuum centrifuge at room temperature for 2 h. Residual water was removed by freeze-drying and the dry extracts were stored at
2 868C until HPLC analysis. 2.5. HPLC analysis of MAAs
Dried extracts of P . antarctica and krill were reconstituted in 200 ml of 100
methanol and left to stand at room temperature for 2 h with vortex mixing at 0.5 h intervals. The re-suspended extracts were centrifuged at 20 000 g to precipitate salts and
other insoluble material. Known volumes of between 3 and 10 ml were injected into a Shimadzu HPLC system utilising a Phenosphere
E 5m C-8 reversed-phase HPLC column 4.6 3 250 mm with a Phenosphere
E 5m C-8 4.6 3 50 mm guard column; the mobile phase consisted of 40 methanol, 59.9 water and 0.1 acetic acid
21
delivered at a flow rate of 0.8 ml min . Integrated peak areas were determined at
detection wavelengths of 313 and 340 nm. MAAs were identified by co-chromatography with prepared standards and comparison
of 313 340 wavelength ratios. Quantified standards were extracted and purified from the following
organisms: the
zoanthid Palythoa
tuberculosa mycosporine-glycine,
palythine, palythinol, the red alga Porphyra tenera shinorine, porphyra-334 and ocular lenses of the coral trout Plectropomus leopardus asterina-330 and palythene. Sec-
ondary calibration of these extract standards was achieved by HPLC analyses from primary calibration of MAAs determined at the Australian Institute of Marine Science
AIMS. Validation and secondary calibration for mycosporine-glycine:valine was conducted at AIMS using an authenticated sample from a dried krill extract prepared
from a previous study at Palmer Station Dunlap et al., 1995. For calculations of MAA concentration, wet mass was converted to approximate dry mass using a water content of
76 Morris et al., 1988.
2.6. Statistical analysis Effect of time and MAA identity on MAA concentration in the P
. antarctica culture was tested using a two-way analysis of variance ANOVA without replication Zar,
1996. The interactive effect of time, MAA identity and algal diet on MAA con- centrations in krill was tested for statistical significance by three-way ANOVA using the
S .J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
99
Systat E 5.01 package for PC Systat. In addition, we performed two-way ANOVAs for
individual MAAs with time and treatment as factors, using StatView E 4.5 Abacus
Concepts. The effect of 36 days of starvation on concentration of individual MAAs in krill, i.e. the difference in mean MAA concentration between day 63 and day 98, was
tested using Student’s t-test Zar, 1996.
3. Results
