Aquaculture 187 Ž2000.

85–96
www.elsevier.nlrlocateraqua-online

Evaluation of diets for culture of the calanoid

copepod Gladioferens imparipes
M.F. Payne ) , R.J. Rippingale
School of EnÕironmental Biology, Curtin UniÕersity of Technology, GPO Box U1987, Perth, Western
Australia 6845, Australia

Accepted 9 December 1999

Abstract

Provision of copepod nauplii as food increases larval survival in many fish species that are
difficult to rear using standard practices. Gladioferens imparipes is a temperate estuarine calanoid
copepod with culture potential. Diets were tested to determine which would maximise copepod
production. Copepods were fed five diets: Isochrysis galbana, Chaetoceros muelleri, Dunaliella
tertiolecta, Nannochloropsis oculata and baker’s yeast. Copepod survival, time to maturity,
maturation rate, nauplius production and female length were recorded at 208C and 258C. By most
criteria, Isochrysis was the most efficacious diet, followed by Chaetoceros and then Dunaliella.
Nannochloropsis and yeast resulted in little or no survival. Fatty acid profile of each diet was
determined. In general, copepod production was positively related to the ratio of DHA:EPA in
their diet. Poor survival on Nannochloropsis was probably a result of the small cell size and poor
digestibility of this species. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: HUFA; Calanoid; Copepod; Algae; Culture; Diet

1. Introduction

For many fish species, hatchery production is uneconomical or impossible using

either rotifers or Artemia as larval diets. This is mostly a result of poor survival at the
first feeding stage. The provision of copepod nauplii in the early larval diet often

)
Corresponding author. Tel.: q61-8-9266-7915; fax: q61-8-9266-2495.
E-mail address: epayne2@cc.curtin.edu.au ŽM.F. Payne..

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 9 9 . 0 0 3 9 1 - 9
86 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96

increases survival in these fish. Species in which this has been recently demonstrated
include red snapper ŽDoi et al., 1994. and some of the groupers ŽDoi et al., 1997.. Also,
the provision of copepods in the early larval diet decreases malpigmentation in halibut
ŽMcEvoy et al., 1998. and increases stress resistance in mahi-mahi ŽKraul et al., 1993..
These improvements are mostly attributed to increased feeding by fish on copepods and
the high nutritional content of copepods.
Copepod culture must demonstrate reliable and prolific production to be accepted as
part of commercial operations. Both calanoid and harpacticoid copepods have been
cultured for use in fish larviculture. Harpacticoids are amenable to high-density culture
conditions, hence, they are the subject of most studies on intensive copepod culture
Že.g., Sun and Fleeger, 1995; Støttrup and Norsker, 1997; Nanton and Castell, 1998..
However, harpacticoid copepods are predominantly benthic and so are not readily
available to fish larvae that feed in the water column ŽKitajima, 1973.. In contrast,
calanoid copepods are almost entirely planktonic, making them more suitable for feeding
to larval fish. However, production from calanoid copepod cultures is typically much
lower than that from harpacticoid cultures ŽStøttrup et al., 1986; Schipp et al., 1999..
Calanoid copepod production is limited by difficulties in maintaining broodstock at
high densities. Stress caused by crowding decreases fecundity in the calanoid Cen-
tropages typicus ŽMiralto et al., 1996.. Also, cannibalism of nauplii by adults reduces
production in dense cultures. Rippingale and MacShane Ž1991. have identified a
temperate estuarine species, Gladioferens imparipes, that exhibits high nauplius produc-
tion at relatively high broodstock densities when cultured on a small scale and does not
consume its own nauplii. While late copepodid and adult stages of this calanoid species
adhere to surfaces, nauplii are entirely pelagic ŽRippingale, 1994..
To increase the scale of G. imparipes cultivation, algal diets that maximise copepod
growth, survival and fecundity must be identified. Diets that are high in highly
unsaturated fatty acids ŽHUFAs. maximise fecundity in the calanoid Acartia tonsa
ŽStøttrup and Jensen, 1990. and are generally the choice of diet for calanoid cultivation
Že.g., Rippingale and MacShane, 1991; Klein Breteler and Laan, 1993; Schipp et al.,
1999.. More specifically, the dietary ratio of two HUFAs, docosahexaenoic acid ŽDHA.
and eicosapentaenoic acid ŽEPA., may affect copepod fecundity ŽStøttrup and Jensen,
1990.
A wide range of algal species from different taxonomic groups are in common use in
aquaculture, each with specific nutritional characteristics. Four algal species from
different groups were fed to copepods in the present work: the prymnesiophyte
Isochrysis galbana ŽT-Iso., the diatom Chaetoceros muelleri, the chlorophyte Dunaliella
tertiolecta and the eustigmatophyte Nannochloropsis oculata. Baker’s yeast Ž Sac-
charomyces cereÕisiae . was also included as a copepod diet. I. galbana, C. muelleri and
N. oculata each have a high HUFA content Žreview by Brown et al., 1997., whereas D.
tertiolecta and yeast contain little or no HUFAs ŽStøttrup and Jensen, 1990; Whyte and
Nagata, 1990..
The present work was planned to determine the diets that will improve production of
the calanoid copepod G. imparipes at two different temperatures using the criteria of
copepod survival, time to maturity, maturation rate, nauplius production and female
prosome length.
 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96 87

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
animals of approximately uniform age class Ž- 24 h old.. These nauplii were mixed
with filtered Ž1.2 mm. seawater diluted to 27‰ to give a density of 1 naupliusrml.
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
I. galbana Ž67 fl 1 ., C. muelleri Ž90 fl., D. tertiolecta Ž153 fl. and N. oculata Ž16 fl. and
fresh baker’s yeast Ž 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 fr2 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
Counter ŽModel D.. Prior to feeding each day, the turbidity of each algal culture was
measured using a Hach Kit ŽDrelr5. 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

Fatty acids were analysed using techniques modified from Dunstan et al. Ž1992.. For
each diet, between 250 and 500 ml of dense cell suspension was concentrated on
Whatman w GFrC filter paper under gentle vacuum. The paper was immersed in
dichloromethane ŽDCM.:methanol:water Ž1:2:0.8 vrvrv; 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
rinsed with DCM:methanol:water Ž50 ml.. Lipids were extracted in DCM from a

1
Mean cell volume in femtolitres Ž10y1 5 l..
88 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96

mixture of DCM:water Ž1:1 vrv.. The solvent was removed using a Bucci w rotary
evaporator and the remaining lipids dissolved in methanol Ž5 ml.. Samples were
transferred to 20 ml reaction tubes, combined with acidified methanol Ž4 ml., toluene Ž2
ml. and an internal standard Žnonadecanoic acid Ž0.5 mg.. and heated to 508C for 16 h.
The cooled reaction mixture was transferred to hexane Ž20 ml., washed twice with
deionised water Ž20 ml. and dried over anhydrous sodium sulphate.
The relative fatty acid methyl ester ŽFAME. composition of these solutions was
determined by gas chromatography ŽGC. with mass selective detection ŽHewlett
Packard w ŽHP. a5890 Series II Gas Chromatograph with a HP a5971 Series Mass
Selective Detector.. The GC was fitted with a DB23 column ŽJ & 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
 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96 89

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 naupliirfemalerday, 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.

3. Results

3.1. Fatty acid analyses of copepod diet

Table 1 shows that Isochrysis contained the greatest proportion of DHA Ž22:6n y 3.,
followed by Chaetoceros. This fatty acid was not detected in the other diets. EPA
Ž20:5n y 3. was the most abundant fatty acid in Nannochloropsis and Chaetoceros.
Isochrysis contained only a small proportion of this fatty acid. The high proportion of
EPA resulted in Nannochloropsis recording the largest proportion of HUFAs, followed
by Chaetoceros and Isochrysis. DHA:EPA ratio was greatest in Isochrysis. In contrast,
Dunaliella and yeast recorded negligible HUFA content. The most abundant fatty acids
in these diets were 18:3n y 3 and 18:1n y 9, respectively.

Table 1
Fatty acid content Ž% of total fatty acids. of diets used to rear copepods. Means of 3 replicates"SD
Fatty acid Isochrysis Chaetoceros Dunaliella Nannochloropsis Yeast
C14:0 15.56"0.42 22.98"1.43 0.4"0.19 4.57"0.38 0.63"0.06
C15:0 0.43"0.03 0.32"0.04 – 0.31"0.02 0.23"0.04
C16:0 13.08"0.53 7.95"0.25 13.48"0.14 17.03"0.4 8.83"0.28
C16:1 6.05"0.44 18.12"0.65 1.72"0.01 16.2"0.28 34.25"4.6
C16:2 0.39"0.03 – 1.16"0.07 0.25"0.05 –
C16:3 – 14.16"0.3 3.96"0.39 – –
C16:4 – – 21.17"0.52 – –
C18:0 0.38"0.06 0.41"0.03 0.72"0.45 0.44"0.08 2.86"0.12
C18:1ny7 2.48"0.06 0.51"0.15 – – 1.85"0.22
C18:1ny9 16.69"0.61 0.63"0.01 – 5.48"0.26 43.39"2.75
C18:2 ny6 2.25"0.23 0.69"0.08 4.22"0.05 1.73"0.03 6.9"1.59
C18:2 ny8 – – 2.24"0.06 – –
C18:3ny3 – – 47.25"0.49 – –
C18:3ny6 – 1.74"0.25 3.2"0.09 0.42"0.08 –
C18:4ny3 26.03"0.74 0.84"0.12 – – –
C20:4ny6 – 3.72"0.14 – 7.58"0.32 –
C20:5ny3 0.31"0.21 25.15"2.65 – 44.26"0.53 –
C22:0 0.1"0.03 – – – –
C22:6ny3 16.2"0.93 2.44"0.49 – – –
C24 – 0.35"0.08 0.43"0.16 1.13"0.69 0.9"0.31
Total HUFA 16.51"0.75 31.31"2.1 tr 52.45"0.49 0
DHA:EPA 52.3 0.1 0 0 0
90 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96

Fig. 1. Copepod Ž G. imparipes . survival Žmean"SD. to maturity fed different diets at 208C and 258C.
Different letters denote a significant difference between values at the same temperature Ž P - 0.05..

3.2. Copepod surÕiÕal

Fig. 1 shows that, at both temperatures, survival was generally greatest on a diet of
Isochrysis, followed by Dunaliella, Chaetoceros and Nannochloropsis. At 208C, sur-
vival was significantly greatest on a diet of Isochrysis and lowest on a diet of
Nannochloropsis. Other results were not significant. Copepods did not survive to
maturity on a diet of Nannochloropsis at 258C or on a diet of yeast at either
temperature. However, some copepods survived 9 days longer than unfed controls on a
diet of yeast at 208C.
3.3. Maturation
Fig. 2 shows that at 208C, the time taken for 50% of female copepods to mature was
significantly shortest with a diet of Isochrysis. Time to maturity increased with a diet of

Fig. 2. Cumulative maturation Žlogit transformed. over time of female copepods Ž G. imparipes. fed Isochrysis
Že., Chaetoceros Ž^., Dunaliella Ž`. and Nannochloropsis Ž\. at 208C. Dotted lines represent time
Ž x-axis. taken for 50% cumulative maturation Ž y-axis.. Days and gradients Ž b 1 . with different superscripts are
significantly different Ž P - 0.05..
 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96 91

Fig. 3. Cumulative maturation Žlogit transformed. over time of female copepods Ž G. imparipes. fed Isochrysis
Že., Chaetoceros Ž^. and Dunaliella Ž`. at 258C. Dotted lines represent time Ž x-axis. taken for 50%
cumulative maturation Ž y-axis.. Days and gradients Ž b 1 . with different superscripts are significantly different
Ž P - 0.05..

Chaetoceros. Copepods fed Dunaliella and Nannochloropsis took the longest time to
mature. The same trend occurred at 258C although copepods matured faster ŽFig. 3..
However, results were not significantly different at this temperature and no copepod
maturation was recorded on a diet of Nannochloropsis.

Fig. 4. G. imparipes nauplius production per female per day and female prosome length Žboth mean"SD. fed
different diets at 208C and 258C. Letters show significance of data at the same temperature, different letters
indicate a significant difference Ž P - 0.05..
92 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96

Copepod maturation rate, as indicated by the regression gradients, was the same for
animals on different diets at 208C ŽFig. 2.. At 258C, this rate was significantly greater
for Isochrysis fed copepods compared to those with the other diets ŽFig. 3..

3.4. Nauplius production and female length

Fig. 4 shows that at both 208C and 258C, daily nauplius production was greatest for
females on a diet of Isochrysis. At 208C, there was no difference in production on the
other diets, whereas at 258C, production on a diet of Chaetoceros was greater than on a
diet of Dunaliella.
Prosome length was greatest for females reared at 208C with a diet of Isochrysis and
least with a diet of Dunaliella. At 258C, copepods fed Isochrysis and Chaetoceros were
longer than those fed Dunaliella. For those copepods fed Isochrysis, Chaetoceros and
Dunaliella, there appeared to be a positive correlation between female prosome length
and nauplius production at each temperature. In these copepods, length and nauplius
production appeared to be determined more by diet than by temperature.

4. Discussion

The fatty acid profiles of algae used in copepod diets compare well with published
data. Table 1 shows that Isochrysis contained the highest proportion of DHA, and had a
high DHA:EPA ratio, as was recorded by Pillsbury Ž1985. and Dunstan et al. Ž1993..
Chaetoceros is rich in EPA and recorded a low DHA:EPA ratio ŽNapolitano et al.,
1990. and Dunaliella is most often recorded as containing little or no HUFAs but a
substantial proportion of 18:3n y 3 ŽPillsbury, 1985; Norsker and Støttrup, 1994.. A
high EPA content in Nannochloropsis is consistent with the findings of Dunstan et al.
Ž1993. and Sukenik et al. Ž1993.. Baker’s yeast contains no HUFAs but large quantities
of the fatty acids 16:1 and 18:1n y 9 ŽWatanabe et al., 1983; Whyte and Nagata, 1990..
Copepod survival ŽFig. 1. and nauplius production ŽFig. 4. were greatest, and time to
maturity ŽFig. 2. shortest with a diet of Isochrysis at 208C. At 258C, nauplius production
ŽFig. 4. was greatest on this diet. Of the experimental diets fed to G. imparipes,
Isochrysis was clearly the most efficacious. This supports the work of Arnott et al.
Ž1986., who recorded high survival to maturity in a similar copepod species, G.
pectinatus, fed this algal species. Further, I. galbana was the only algal diet that
sustained viable egg production through multiple generations at 188C. In contrast, D.
tertiolecta did not sustain viable egg production in this species. These workers specu-
lated that Isochrysis contained essential micronutrients not present in Dunaliella. There
is now abundant evidence that these micronutrients include HUFAs, particularly DHA.
Støttrup and Jensen Ž1990. examined egg production in the calanoid A. tonsa fed
five different microalga. As in the present work, they recorded the greatest egg
production on a diet of I. galbana and comparatively low production on D. tertiolecta.
These workers observed no relationship between dietary HUFA content and fecundity,
although there was a correlation between DHA:EPA ratio and egg production, with the
highest ratio supporting the highest egg production. This correlation was also observed
 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96 93

in A. tonsa by Jonasdottir and Kiørboe Ž1996.. A similar trend was recorded in the
current study, suggesting that the DHA:EPA ratio is a more important criterion than
HUFA content when selecting a diet for maximising egg production in calanoid
copepods.
After Isochyrsis, Chaetoceros was the next most beneficial diet for G. imparipes.
Small quantities of DHA in this alga resulted in a lower DHA:EPA ratio than recorded
in Isochrysis ŽTable 1.. Copepods fed Chaetoceros tended to have a shorter time to
maturity and produce more nauplii than those fed Dunaliella. Despite this, survival at
both temperatures tended to be lower on this diet compared to Dunaliella, although not
by a significant margin. There is conflicting evidence that diatoms such as Chaetoceros
have an inhibitory effect on calanoid copepod reproduction ŽIanora et al., 1995;
Jonasdottir and Kiørboe, 1996.. While inhibition of reproduction was not observed in G.
imparipes, the value of this alga Žor another diatom. as a monodiet for intensive culture
requires further investigation.
By most criteria, Dunaliella was a less efficacious diet for G. imparipes than
Isochrysis in this study. Dunaliella contained no DHA, hence a DHA:EPA ratio could
not be calculated ŽTable 1.. This is consistent with the findings of Støttrup and Jensen
Ž1990. mentioned previously. Also, Arnott et al. Ž1986. found that, unlike Isochrysis,
Dunaliella did not sustain viable egg production in G. pectinatus.
No survival to maturity was observed in G. imparipes fed yeast. Like Artemia
ŽCoutteau et al., 1990., copepods may be unable to fully digest yeast cells. Some
copepods survived longer than the unfed controls at 208C, but not at 258C. At lower
temperatures, copepod growth is depressed ŽKlein Breteler and Schogt, 1994. as a result
of lower metabolic activity. It is possible that G. imparipes received enough energy
from yeast to sustain metabolism for a short time at 208C. The mean volume of yeast
cells was comparable to that of the three most successful diets in this trial, Isochrysis,
Chaetoceros and Dunaliella. Hence, nauplii should have been able to ingest yeast.
Despite poor copepod survival, yeast may still have some potential as an indirect
food for G. imparipes. Yeast can be used effectively to culture dinoflagellates ŽKleppel
and Burkart, 1995. and G. imparipes will readily consume some species of dinoflagel-
late ŽS. Griffin, pers. comm... Alternatively, dead yeast cells provide substrate for
bacteria and some calanoid copepod nauplii can utilise bacteria as a food source ŽRoff et
al., 1995.. Either dinoflagellates or bacteria could have been available to copepods as
food in yeast-fed treatments and may have contributed to limited survival of G.
imparipes at 208C. Some calanoid copepods exhibit high rates of growth and fecundity
when fed dinoflagellates compared to microalgae or yeast alone ŽKleppel and Burkart,
1995; Klein Breteler et al., 1999., hence this requires investigation in G. imparipes.
A diet of Nannochloropsis resulted in no copepod survival to maturity at 258C ŽFig.
1. and reduced survival, development and egg production at 208C ŽFigs. 1, 2 and 4..
Given the high HUFA content of this alga, this result is unexpected. However, the mean
cell volume of Nannochloropsis was considerably smaller than cells of the other diets
fed to copepods. This may make it too small for G. imparipes nauplii to capture from
the water column. Also, Nannochloropsis has a hard cell wall that makes it indigestible
to Artemia ŽDhont and Lavens, 1996. and, hence perhaps to G. imparipes as well. As
with a yeast diet, copepods probably received some direct or indirect nutrition from this
94 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96

alga and lower metabolic demands at a lower temperature resulted in limited survival at
208C. This prolific and nutritious alga is in common use in aquaculture as a diet for
rotifers and its use in the mass culture of G. imparipes should not be discounted.
Female length appeared to correlate more with diet than with temperature in G.
imparipes ŽFig. 4.. Thus, copepod growth on Chaetoceros, Dunaliella and Nan-
nochloropsis was nutrient-limited. Fig. 4 also shows a positive correlation between
female length and nauplius production. This relationship has been well established for
G. imparipes ŽRippingale and Hodgkin, 1974. and other calanoid copepods ŽMcLaren,
1965.. Shortened time to maturity ŽFig. 3. and increased nauplius production on the two
most successful diets ŽFig. 4. at 258C show that culture production would be maximised
at this higher temperature.
Nutrition is such a complex issue that it is simplistic to consider one dietary factor in
isolation. Components other than HUFAs are also important to copepod health and
fecundity. Dietary content of essential amino acids has been correlated to egg production
in A. tonsa by Kleppel et al. Ž1998.. Also, these workers recorded poor egg production
in copepods fed with a strain of Isochrysis that was found to be unusually deficient in
the amino acid histidine. This suggests that copepod amino acid requirements may be at
least as important as their HUFA requirements and thus requires more attention.
This study sought to determine diets that improve production of G. imparipes in
intensive culture. However, the nutritional adequacy of these copepods for use as food
for larval fish has yet to be assessed. Feeding G. imparipes broodstock with a high
HUFA diet such as I. galbana will be likely to result in HUFAs being incorporated in
the lipid reserves of nauplii. Hence, these nauplii should be nutritious food for larval fish
without the need for post-harvest enrichment.

Acknowledgements

Financial support was received from the Fisheries Research and Development
Corporation ŽProject No. 96r398..

References

Arnott, G.H., Brand, G.W., Kos, L.C., 1986. Effects of food quality and quantity on the survival, development,
and egg production of Gladioferens pectinatus ŽBrady. ŽCopepoda: Calanoida.. Aust. J. Mar. Freshwater
Res. 37, 467–473.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem.
Physiol. 37, 912–917.
Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, G.A., 1997. Nutritional properties of microalgae for
mariculture. Aquaculture 151, 315–331.
Coutteau, P., Lavens, P., Sorgeloos, P., 1990. Baker’s yeast as a potential substitute for live algae in
aquaculture diets: Artemia as a case study. J. World Aquacult. Soc. 21, 1–9.
Dhont, J., Lavens, P., 1996. Tank production and use of ongrown Artemia. In: Lavens, P., Sorgeloos, P.
ŽEds.., Manual on the Production and Use of Live Food for Aquaculture. FAO Fisheries Technical Paper
No. 361, FAO, Rome, pp. 164–195.
 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96 95

Doi, M., Singhagraiwan, T., Singhagraiwan, S., Ohno, A., 1994. An investigation of copepods being applied
as initial food organisms for red snapper larvae. Thai Mar. Fish. Res. Bull. 5, 21–26.
Doi, M., Toledo, J.D., Golez, M.S.N., Santos, M.d.l., Ohno, A., 1997. Preliminary investigation of feeding
performance of larvae of early red-spotted grouper, Epinephelus coioides, reared with mixed zooplankton.
Hydrobiologia 358, 259–263.
Dunstan, G.A., Volkman, J.K., Barrett, S.M., Garland, C.D., 1993. Changes in the lipid composition and
maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture. J. Appl.
Phycol. 5, 71–83.
Dunstan, G.A., Volkman, J.K., Jeffrey, S.W., Barrett, S.M., 1992. Biochemical composition of microalgae
from the green algal classes Chlorophyceae and Prasinophyceae: 2. Lipid classes and fatty acids. J. Exp.
Mar. Biol. Ecol. 161, 115–134.
Ianora, A., Poulet, S.A., Miralto, A., 1995. A comparative study of the inhibitory effect of diatoms on the
reproductive biology of the copepod Temora stylifera. Mar. Biol. 121, 533–539.
Jonasdottir, S.H., Kiørboe, T., 1996. Copepod recruitment and food composition: do diatoms affect hatching
success? Mar. Biol. 125, 743–750.
Kitajima, C., 1973. Experimental trials on mass culture of copepods. Bull. Plankton Soc. Jpn. 20, 54–60.
Klein Breteler, W.C.M., Laan, M., 1993. An apparatus for automatic counting and controlling density of
pelagic food particles in cultures of marine organisms. Mar. Biol. 116, 169–174.
Klein Breteler, W.C.M., Schogt, N., 1994. Development of Acartia clausi ŽCopepoda, Calanoida. cultured at
different conditions of temperature and food. Hydrobiologia 292r293, 469–479.
Klein Breteler, W.C.M., Schogt, N., Bass, M., Schouten, S., Kraay, G.W., 1999. Trophic upgrading of food
quality by protozoans enhancing copepod growth: role of essential lipids. Mar. Biol. 135, 191–198.
Kleppel, G.S., Burkart, C.A., 1995. Egg production and the nutritional environment of Acartia tonsa: the role
of food quality in copepod nutrition. ICES J. Mar. Sci. 52, 297–304.
Kleppel, G.S., Burkart, C.A., Houchin, L., 1998. Nutrition and the regulation of egg production in the calanoid
copepod Acartia tonsa. Limnol. Oceanogr. 43, 1000–1007.
Kraul, S., Brittain, K., Cantrell, R., Nagao, T., Ako, H., Ogasawara, A., Kitagawa, H., 1993. Nutritional
factors affecting stress resistance in the larval mahimahi Coryphaena hippurus. J. World Aquacult. Soc.
24, 186–193.
McEvoy, L.A., Naess, T., Lie, O., 1998. Lipid and fatty acid composition of normal and malpigmented atlantic
halibut Ž Hippoglossus hippoglosus. fed enriched Artemia: a comparison with fry fed wild copepods.
Aquaculture 163, 237–250.
McLaren, I.A., 1965. Some relationships between temperature and egg size, body size, development rate and
fecundity of the copepod Pseudocalanus. Limnol. Oceanogr. 10, 528–538.
Miralto, A., Ianora, A., Poulet, S.A., Romano, G., Laabir, M., 1996. Is fecundity modified by crowding in the
copepod Centropages typicus? J. Plankton Res. 18, 1033–1040.
Nanton, D.A., Castell, J.D., 1998. The effects of dietary fatty acids on the fatty acid composition of the
harpacticoid copepod, Tisbe sp., for use as a live food for marine fish larvae. Aquaculture 163, 251–261.
Napolitano, G.E., Ackman, R.G., Ratnayaye, W.M.N., 1990. Fatty acid composition of three cultured algal
species Ž Isochrysis galbana, Chaetoceros gracilis and C. calcitrans. used as food for bivalve larvae. J.
World Aquacult. Soc. 21, 122–130.
Norsker, N.H., Støttrup, J.G., 1994. The importance of dietary HUFAs for fecundity and HUFA content in the
harpacticoid, Tisbe holothuriae Humes. Aquaculture 125, 155–166.
Pillsbury, K.S., 1985. The relative food value and biochemical composition of five phytoplankton diets for
Queen Conch, Strombus gigas ŽLinne. larvae. J. Exp. Mar. Biol. Ecol. 90, 221–231.
Rippingale, R.J., 1994. A calanoid copepod Gladioferens imparipes, holding to surfaces. Hydrobiologia
292r293, 351–360.
Rippingale, R.J., Hodgkin, E.P., 1974. Population growth of a copepod Gladioferens imparipes Thomson.
Aust. J. Mar. Freshwater Res. 25, 351–360.
Rippingale, R.J., MacShane, M.G., 1991. A calanoid copepod for intensive cultivation. Mem. Queensl. Mus.
31, 457.
Roff, J.C., Turner, J.T., Webber, M.K., Hopcroft, R.R., 1995. Bacterivory by tropical copepod nauplii: extent
and possible significance. Aquat. Microb. Ecol. 9, 165–175.
96 M.F. Payne, R.J. Rippingaler Aquaculture 187 (2000) 85–96

Schipp, G.R., Bosmans, J.M.P., Marshall, A.J., 1999. A method for hatchery culture of tropical calanoid
copepods, Acartia spp. Aquaculture 174, 81.
Støttrup, J.G., Jensen, J., 1990. Influence of algal diet on feeding and egg-production of the calanoid copepod
Acartia tonsa Dana. J. Mar. Biol. Assoc. U. K. 141, 87–105.
Støttrup, J.G., Norsker, N.H., 1997. Production and use of copepods in marine fish larviculture. Aquaculture
155, 231–248.
Støttrup, J.G., Richardson, K., Kirkegaard, E., Pihl, N.J., 1986. The cultivation of Acartia tonsa Dana for use
as a live food source for marine fish larvae. Aquaculture 52, 87–96.
Sukenik, A., Zmora, O., Carmeli, Y., 1993. Biochemical quality of marine unicellular algae with special
emphasis on lipid composition: II. Nannochloropsis sp. Aquaculture 117, 313–326.
Sun, B., Fleeger, J.W., 1995. Sustained mass culture of Amphiascoides atopus a marine harpacticoid copepod
in a recirculating system. Aquaculture 136, 313–321.
Watanabe, T., Kitajima, C., Fujita, S., 1983. Nutritional values of live organisms used in Japan for mass
propagation of fish: a review. Aquaculture 34, 115–143.
Whyte, J.N.C., Nagata, W.D., 1990. Carbohydrate and fatty acid composition of the rotifer, Brachionus
plicatilis, fed monospecific diets of yeast or phytoplankton. Aquaculture 89, 263–272.