2. Materials and method
2.1. General methods Nauplii were collected from 30-l copepod broodstock cultures maintained on a diet of
I. galbana at 228C and 27‰ salinity. Screening nauplii through 100 mm mesh provided Ž
. animals of approximately uniform age class - 24 h old . These nauplii were mixed
Ž .
with filtered 1.2 mm seawater diluted to 27‰ to give a density of 1 nauplius rml.
Nauplii were then randomly allocated to each of 36 150-ml plastic containers such that each contained approximately 80 nauplii. Volumes were made up with filtered seawater
diluted to 27‰. Two groups of 18 containers each were placed in separate water-baths maintained at 20 0.58C and 25 0.58C. Lids on the water-baths shaded the containers
from direct light. Five treatment diets were each randomly allocated to three containers in both groups, with the remaining three left unfed. Diets consisted of the algal species
Ž
1
. Ž
. Ž
. Ž
. I. galbana 67 fl
, C. muelleri 90 fl , D. tertiolecta 153 fl and N. oculata 16 fl and Ž
. fresh baker’s yeast S. cereÕisiae; 83 fl . Hereafter, algal species will be referred to by
their generic name. Algal cultures were originally obtained from the CSIRO Marine Laboratories in Hobart, Tasmania, as non-axenic cultures, and maintained as batch
cultures in 5-l Pyrex
w
flasks using Guillard’s f r2 medium. Algae was fed to copepods
only when in mid to late logarithmic phase. Nauplii were fed daily with equivalent concentrations by volume of the treatment
diets. The mean cell volumes of each food organism were determined using a Coulter Ž
. Counter Model D . Prior to feeding each day, the turbidity of each algal culture was
Ž .
measured using a Hach Kit Drel r5 and used to calculate cell density. The correlation
equation used in this process was recalculated weekly. Suspensions of fresh yeast in deionised water were stored at 48C and renewed every 2 days. A haemocytometer was
used to determine cell densities of each new suspension. Cell volume and density were then used to calculate a quantity of food such that treatments received 1 ppm food by
volume each day up to day 4, increasing to 2 ppm for the duration of the trials. These quantities ensured excess food in all treatments, as indicated by the presence of uneaten
algal or yeast cells in containers at the next feed. Every third day, 90 of the volume in each container was replaced with clean water.
2.2. Fatty acid analyses of copepod diets Ž
. Fatty acids were analysed using techniques modified from Dunstan et al. 1992 . For
each diet, between 250 and 500 ml of dense cell suspension was concentrated on Whatman
w
GF rC filter paper under gentle vacuum. The paper was immersed in
Ž .
Ž .
dichloromethane DCM :methanol:water 1:2:0.8 v rvrv; Bligh and Dyer, 1959 , mashed
with a glass rod and subject to ultrasonification for 10 min. It was then covered and stored in a refrigerator overnight. This sample was filtered under gentle vacuum and
Ž .
rinsed with DCM:methanol:water 50 ml . Lipids were extracted in DCM from a
1
Ž
y1 5
. Mean cell volume in femtolitres 10
l .
Ž .
w
mixture of DCM:water 1:1 v rv . The solvent was removed using a Bucci
rotary Ž
. evaporator and the remaining lipids dissolved in methanol
5 ml . Samples were Ž
. Ž
transferred to 20 ml reaction tubes, combined with acidified methanol 4 ml , toluene 2 .
Ž Ž
.. ml and an internal standard nonadecanoic acid 0.5 mg
and heated to 508C for 16 h. Ž
. The cooled reaction mixture was transferred to hexane 20 ml , washed twice with
Ž .
deionised water 20 ml and dried over anhydrous sodium sulphate. Ž
. The relative fatty acid methyl ester FAME composition of these solutions was
Ž .
Ž determined by gas chromatography
GC with mass selective detection
Hewlett
w
Ž .
Packard HP a5890 Series II Gas Chromatograph with a HP a5971 Series Mass
. Ž
Selective Detector . The GC was fitted with a DB23 column J W, 30 m = 0.25 mm .
i.d.= 0.25 mm film thickness with helium at 25 kPa used as the carrier gas. Each analytical run commenced with 5 min at 508C, increasing 58C each minute up to 2408C,
with this temperature being maintained for the final 5 min. Retention times were determined relative to that of the internal standard and identification of the FAMEs was
carried out by the comparison of the mass spectral analysis of individual eluted peaks with the Wiley electronic database. Identification of EPA and DHA was confirmed by
comparisons to authentic FAME standards.
2.3. Copepod surÕiÕal Copepod survival was assessed daily using a stereo microscope until egg production
commenced. Containers in which - 10 copepods survived were considered to have no copepod survival and were removed from the trial. Adult copepods were counted at the
conclusion. Results were analysed using one-way ANOVAs and Tukey’s W procedure.
2.4. Maturation Female copepods were judged to be mature when carrying their first clutch of
embryos. Newly mature females were removed individually from containers daily using a stereo microscope and pipette. Females were counted and placed in a separate
container with other females from the same treatment. Daily collection continued until all females had reached maturity and had been removed from the experimental contain-
ers. Males were not removed from the containers as they reached maturity. Remaining males were counted and all males from the three replicates of each treatment pooled.
Pooled males and females continued to be fed the treatment diets.
Cumulative proportions of mature females reared on treatment diets were logit transformed and regressed against time. The time taken for 50 of females to reach
maturity on each diet was predicted from these regressions and compared using one-way ANOVAs and Tukey’s W procedure. Maturation rate was defined as the time between
the collection of the first and last mature female, with a shorter time indicating a faster rate and resulting in a regression line with a steeper gradient. Gradients were compared
using dummy variables.
2.5. Nauplius production and female growth Pooled males and females, which had come to maturity and subsequently been fed on
each treatment diet, were mixed together and fed the treatment diets for 4 days. Three
males and three embryo-bearing females were transferred to each of four containers and fed treatment diets to excess. Survival of the adult copepods was monitored daily. After
exactly 96 h, nauplii and adult copepods were preserved for later enumeration. Produc- tions, expressed as nauplii
rfemalerday, were compared using one-way ANOVAs and Tukey’s W procedure.
Prosome length of females used in the nauplius production trial were measured using an ocular graticule. Results were analysed in the same manner as above.
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