Aquaculture 201 Ž2001.

251–262
www.elsevier.comrlocateraqua-online

Effects of salinity, cold storage and enrichment on

the calanoid copepod Gladioferens imparipes
M.F. Payne, R.J. Rippingale )
Department of EnÕironmental Biology, School of Resource Science and Technology,
Curtin UniÕersity of Technology, GPO Box U1987, Perth, 6845, Australia

Received 16 August 2000; accepted 5 March 2001

Abstract

Intensive culture techniques have been developed for the calanoid copepod Gladioferens
imparipes. Despite reliable, long-term production of nauplii from these cultures, further improve-
ments are required to maximise the number and nutritional content of nauplii available for feeding
to larval fish. The effects of salinity on copepod production and prolonged algal enrichment on
nutritional content of nauplii is shown. Use of cold storage for accumulating large numbers of
nauplii and storing adults with minimum maintenance is evaluated. The size range of G.
imparipes life history stages, particularly early nauplii, is determined. Maximum copepod
production was obtained in salinities of 18–27‰. Fatty acid content of nauplii increased with
prolonged enrichment. Copepod survival was poor at 48C. At 88C, nauplius survival was 99%
after 12 days and adult survival was 90% and 68% after 21 and 42 days, respectively. Early
nauplii were 67 mm in width, similar to nauplii used to rear larvae with small gapes at first
feeding. Implications of these results to the use of G. imparipes as food for fish larvae is
discussed. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Calanoid; Copepod; Culture; Salinity; HUFA; Storage; Enrichment

1. Introduction

Copepod culture is an important component of larviculture techniques for particular

marine finfish species. Provision of copepods has improved larval survival in halibut

)
Corresponding author. Tel.: q61-8-9266-7922; fax: q61-8-9266-2495.
E-mail address: R.Rippingale@info.curtin.edu.au ŽR.J. Rippingale..

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 6 0 9 - 3
252 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262

ŽShields et al., 1999., mahimahi ŽKraul et al., 1992. and grouper ŽToledo et al., 1999..
For species of fish in which the larvae have a small gape at first feeding, particularly the
groupers, small copepod nauplii are an accessible food. A high rate of feeding on
copepod nauplii by these fish larvae leads to improved health and survival of larvae. In
calanoid copepod nauplii, the body width is less that the length. The width is probably
the critical dimension that determines the availability of a nauplius to predation by a fish
larva.
Copepods may be highly nutritious for fish larvae ŽWatanabe et al., 1983., especially
if they are rich in the essential fatty acids docosahexaenoic acid ŽDHA; 22:6n y 3.,
eicosapentaenoic acid ŽEPA; 20:5n y 3. and arachidonic acid ŽAA; 20:4n y 6.. Effica-
cious diets for the larvae of a variety of fish species generally contain DHA and EPA in
the ratio of approximately 2:1. EPA:AA ratio requirements are more varied between fish
species ŽSargent et al., 1999..
Reliable production of large numbers of copepods is difficult. Large outdoor tanks or
ponds can provide copepod numbers sufficient for larval rearing ŽOhno and Okamura,
1988.. However, these require substantial space and lack of control over culture
conditions results in variable copepod production. Intensive copepod culture systems do
not require as much space and allow greater control of the culture environment. Reliable
production is a feature of intensive copepod cultures ŽSun and Fleeger, 1995; Støttrup
and Norsker, 1997; Schipp et al., 1999.. Despite recent developments, production from
large scale copepod cultures is still less than from rotifer cultures of equivalent volume.
The latter currently forms the basis of most commercial larviculture practises. Further
refinements to culture techniques may improve copepod production.
The problem of supplying larvae with large numbers of copepod nauplii may be
partly overcome by developing suitable nauplius storage techniques. Nauplii collected
daily from intensive cultures could be accumulated over a period of time prior to feeding
to larvae. Enriched Artemia nauplii are routinely stored at low temperatures and the
effect of cold storage on the calanoid copepod Acartia tonsa ŽStøttrup et al., 1999. has
been examined. However, in both cases, the aim has been to prevent loss of nutritional
value prior to use as live food for fish larvae.
Intensive culture techniques have been developed for the calanoid copepod Glad-
ioferens imparipes ŽPayne and Rippingale, in press.. These cultures provide relatively
large numbers of nauplii daily over periods exceeding 1 year. This temperate estuarine
copepod has been used to improve rearing of West Australian dhufish larvae ŽPayne et
al., 2001. and seahorse juveniles ŽPayne and Rippingale, 2000b.. As an estuarine
species, G. imparipes is tolerant to a wide range of salinities ŽRippingale and Hodgkin,
1974.. However, the salinity at which production from intensive cultures is maximised is
not known. Also, the effect of enrichment period on fatty acid content of G. imparipes
nauplii has not been determined.
This study complements previous work on the provision of G. imparipes nauplii
from intensive culture to marine fish larvae. Three trials are described in order to
determine the effects of Ža. salinity on copepod survival, growth and fecundity, Žb. cold
storage on copepod survival, and Žc. enrichment period on copepod fatty acid content.
Physical dimensions of a single cohort of G. imparipes are also recorded.
 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262 253

2. Material and methods

2.1. Copepod length and width

Copepod nauplii were collected from 500 l intensive cultures and screened through
100 mm screen to obtain small nauplii of a uniform size class Žlength 116–144 mm;
width 58–77 mm.. These nauplii were stocked into three 2-l containers containing
diluted seawater Ž18‰. maintained at 25 " 0.58C. Length and width of 10 copepods
Žselected at random. from each container were measured daily. Only the prosome length
and width was measured in copepodid stages. Copepods were fed daily with excess
quantities of the alga Isochrysis galbana ŽT-Iso; CSIRO culture code CS-177..

2.2. Salinity

Salinity trials were conducted using copepod nauplii collected from 500 l intensive
cultures Žmaintained at 18‰. and grown to maturity at treatment salinities of 9‰, 18‰,
27‰ and 35‰. These copepods, and those used in subsequent maturation and nauplius
production trials, were fed daily with excess I. galbana ŽPayne and Rippingale, 2000a..
Algae were cultured at approximately 22‰ to minimise osmotic shock to cells added to
copepod cultures at various salinity treatments. In all salinity trials, copepods were
maintained at 25 " 0.58C.

2.2.1. StarÕation surÕiÕal

For each salinity treatment, approximately 50 acclimated adult copepods were
distributed to each of three replicate 150 ml vessels containing clean water at the
corresponding salinity. No food was added to these containers. Dead copepods were
removed and counted twice daily.
Cumulative proportions of copepod mortality at treatment salinities were logit
transformed and regressed against time. Copepod survival time was determined as the
time taken for 50% of copepods to starve and for each treatment salinity, this was
predicted from the regression. Survival times in response to salinity were compared
using one-way ANOVAs and Tukey’s W procedure.

2.2.2. Maturation time

Nauplii collected from adults acclimated to the treatment salinities were passed
through 100 mm screen to give a small size class. For each treatment salinity,
approximately 100 nauplii were stocked into four replicate 150 ml vessels containing
clean water at the corresponding salinity.
Methods for removal of ovigerous females are fully described by Payne and
Rippingale Ž2000a.. Female copepods were judged to be mature when carrying their first
clutch of embryos. Each day, newly matured females were removed from the containers,
counted, and placed in a separate container with other females from the same treatment.
254 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262

Daily collection continued until all females had been removed from the experimental
containers. Males were not removed from treatments. Cumulative proportions of mature
females reared at treatment salinities were logit transformed and regressed against time.
The time taken for 50% of females to reach maturity at each salinity was predicted from
these regressions and compared using one-way ANOVAs and Tukey’s W procedure.

2.2.3. Nauplius production

As for maturation, determination of nauplius production is fully described by Payne
and Rippingale Ž2000a.. When all females had matured, remaining males and collected
females were combined and maintained at the corresponding treatment salinity for 4
days. Three males and three ovigerous females were transferred to each of six vessels
containing clean water at the corresponding treatment salinity. Survival of the adult
copepods was monitored daily. After exactly 96 h, nauplii and adult copepods were
preserved for later enumeration. Nauplius productions, expressed as naupliirfemalerday,
were compared using a one-way ANOVA.

2.3. Cold storage

Copepods of the same size class were grown to maturity in sea water diluted to 18‰
with deionised water. These animals were fed daily with I. galbana to excess and
maintained at approximately 258C. Several days after ovigerous females became preva-
lent, adults were separated from nauplii by screening through 149 mm screen and the
water volume reduced by siphoning through a submerged 44 mm screen. The nauplii
and the adult copepods were then placed separately in clean sea water Ž18‰. and fed
excess I. galbana for 2 h. Following this feeding period, copepods were again
concentrated with 44 mm screen and placed in clean sea water Ž18‰.. Copepods were
distributed evenly between twelve 2-l containers, six containers each for nauplii and
adult copepods. Nauplii and adults were stocked at densities of 2rml and 0.25rml,
respectively. From these, three nauplius containers and three adult copepod containers
were randomly selected and placed into a refrigerator set at 4 " 1.58C ŽWestinghouse w
Silhoutte 141.. Those remaining were placed into a refrigerator set at 8 " 1.58C
ŽWestinghouse w 142.. Lids were placed loosely on each container and no aeration was
provided.
The effect of spatial temperature differences within each refrigerator was reduced by
daily rotation of container positions. Containers were sub-sampled at 3-day intervals by
removing three consecutive 40-ml volumes during thorough gentle mixing of the
contents of each container. Each 120-ml sub-sample was then left to attain room
temperature Ž; 208C. before a small quantity of I. galbana was added. After 24 h, the
number of living and dead copepods in each sub-sample was determined. Living nauplii
and adult copepods sub-sampled on days 21 and 42 were placed in clean sea water
Ž18‰.. These animals were fed daily until live nauplii of the next generation were
observed.
Cumulative proportions of copepod mortality were logit transformed and gradients of
the resulting linear regressions compared using dummy variables. Gradients representing
 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262 255

mortality at 48C and 88C were compared separately for adults and nauplii at these
temperatures.

2.4. Nauplius enrichment

Nauplii were collected from 500 l intensive cultures and placed in clean diluted
seawater Ž18‰. for 24 h. Approximately 17% of the nauplii were collected for
immediate fatty acid analysis. A further 17% of the nauplii were placed in a separate
container and provided a combination of I. galbana and Nannochloropsis oculata
ŽCSIRO culture code CS-179. at densities of 5 = 10 5 and 3 = 10 6 cellsrml, respec-
tively. Remaining nauplii were fed I. galbana at the rate of 5 = 10 5 cellsrml. In each
treatment, nauplii were stocked at approximately 200rml and subjected to gentle
aeration at 238C. Nauplii provided with I. galbana and N. oculata were collected for
fatty acid analysis after 6 h. Those provided I. galbana were collected for analysis after
0.5, 2, 4 and 6 h.
For each analysis, approximately 200,000 nauplii were collected on 50 mm diameter
discs of 44 mm nylon screen. Fatty acids were analysed using techniques modified from
Dunstan et al. Ž1992.. Animals were rinsed into a reaction tube with dichloromethane
ŽDCM.:methanol:water Ž15 ml. and the sample homogenised and stored under nitrogen
at 5–68C for 12 h. Samples were filtered under gentle vacuum and rinsed with
DCM:methanol:water Ž50 ml.. Lipids were extracted in DCM from a mixture of
DCM:water Ž1:1 vrv.. The solvent was removed using a Bucci w rotary evaporator and
the remaining lipid 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 Žnonadecaenoic acid; 0.5 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; Hewlett Packard w ŽHP. a5890.. The GC was
fitted with a HP Innowax column Ž30 m = 0.25 mm i.d.= 0.5 mm film thickness..
Temperature programming for each analytical run was as follows; increased from 1808C
to 2008C at 58Crmin, held at 2008C for 3 min, increased to 2508C at 28Crmin and held
at this temperature for 18 min. Retention times were determined relative to that of the
internal standard and a mixture of FAME standards ŽSigmaw a189-19..

3. Results

3.1. Copepod length and width

Fig. 1 shows the increase in length and width of a cohort of G. imparipes from the
N1 development stage. The average dimensions of early G. imparipes nauplii are
126Žl. = 67Žw. mm.
256 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262

Fig. 1. Length and width Žmm; mean 3 reps"s.d.. of a cohort of G. imparipes Žstocked at the N1 development
stage. reared on I. galbana at 258C. Daily measurements were of 10 copepods that were not all at the same
stage of development. Prosome dimensions of copepodid stages were recorded.

3.2. Salinity

Unfed adult G. imparipes survived longest Ž P - 0.001. when maintained at salinities

of 18‰ and 27‰ ŽFig. 2.. Copepods survived for a longer period Ž P - 0.001. at a
salinity of 9‰ compared to 35‰.
Maturation time was significantly shorter Ž P - 0.001. for those copepods maintained
at a salinity of 27‰ compared with those maintained at 9‰ ŽFig. 3.. There were no
other significant differences. At 27‰, maturation time was 10.4 days.
Salinity had no significant effect on nauplius production. An average nauplius
production of 17.1 " 2.6 naupliirfemalerday was recorded for all salinity treatments.

Fig. 2. Survival of unfed adult G. imparipes maintained at different salinities. Cumulative mortality Žexpressed
as proportions. were logit transformed Ž y-axis. and the day Ž x-axis. on which 50% cumulative mortality
occurred was predicted. Days with different superscripts are significantly different Ž P - 0.001..
 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262 257

Fig. 3. Maturation time of G. imparipes fed I. galbana at different salinities. Cumulative maturation
Žexpressed as proportions. were logit transformed Ž y-axis. and the day Ž x-axis. on which 50% cumulative
maturation occurred was predicted. Days with different superscripts are significantly different Ž P - 0.001..

3.3. Cold storage

For both nauplii and adults, mortality of animals stored at 88C occurred at signifi-
cantly slower rates Ž P - 0.001. compared to those stored at 48C ŽFig. 4.. At 88C,
nauplius survival remained above 99% for the first 12 days of storage before declining.
Survival of adult copepods stored at this temperature was 90% and 68% after 21 and 42
days, respectively. Living nauplii removed from 88C storage on both days 21 and 42
grew to maturity and produced healthy nauplii. Living adults removed from 48C and 88C
storage on both days 21 and 42 also produced healthy nauplii.

3.4. Nauplius enrichment

In G. imparipes nauplii enriched with I. galbana only, DHA content increased and
EPA content slightly decreased after 4 h of enrichment ŽTable 1.. After 6 h, DHA:EPA
ratio increased to a maximum of 7.0 in these nauplii. In general, total fatty acid content

Fig. 4. Survival of G. imparipes nauplii and adults maintained at 48C and 88C. Mean 3 reps"s.d.
258 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262

Table 1
Fatty acid content ŽmgrgDW; 1 gDWs9.14=10 6 nauplii. of G. imparipes nauplii enriched with I. galbana
for between 0 and 6 h and with a combination of I. galbana and N. oculata for 6 h. Mean of 3 reps"s.d.
Fatty acid Enriched with I. galbana I. galbana and N. oculata
0h 0.5 h 2h 4h 6h 6h
14:0 3.0"0.4 4.6"0.6 5.1"0.8 5.1"1.0 5.6"1.4 6.0"0.9
16:0 11.8"2.1 12.3"1.9 11.2"1.5 10.0"1.2 11.3"1.3 13.4"1.6
16:1 1.8"0.7 1.5"0.3 0.8"0.7 1.2"0.3 1.2"0.3 1.8"0.4
18:0 2.9"1.0 2.3"0.6 1.9"0.2 1.6"0.1 1.5"0.0 2.4"1.2
18:1 9.6"2.8 10.2"2.5 9.1"1.0 9.0"2.4 8.2"2.4 11.3"2.0
18:2 1.5"0.1 2.3"0.4 2.3"0.4 2.5"0.5 2.4"0.6 2.7"0.4
18:3ny3 0.7"0.1 1.2"0.2 1.3"0.3 1.6"0.3 1.7"0.3 1.7"0.3
18:4ny3 1.8"0.7 3.2"0.7 3.4"0.6 4.1"0.8 4.4"1.1 4.5"0.7
20:2 1.4"0.2 1.0"0.2 1.2"0.5 1.5"0.3 1.2"0.3 1.8"0.7
20:4ny6 nd nd nd nd nd 0.9"0.4
20:5ny3 1.4"0.4 1.4"0.3 1.4"0.2 1.3"0.1 1.3"0.1 2.8"0.2
22:6ny3 6.9"0.2 7.8"0.1 7.9"1.2 8.6"0.6 9.1"0.2 10.1"0.9
Ýfatty acid 42.9"6.6 48.0"7.2 45.7"5.5 46.5"6.6 47.9"7.4 59.5"6.6
DHA:EPA 4.9 5.6 5.6 6.6 7.0 3.6
EPA:AA – – – – – 3.1

nd indicates not detected.

– indicates cannot be calculated.

also increased over this period. AA was not detected in nauplii enriched with I. galbana
only. Nauplii enriched with a mixture of I. galbana and N. oculata for 6 h contained
AA and a higher level of EPA relative to DHA. DHA:EPA and EPA:AA ratios of 3.6
and 3.1, respectively, and total fatty acid content of 59.5 mgrgDW were recorded in
these nauplii.

4. Discussion

Early G. imparipes nauplii are similar in width to nauplii of other copepod species
that have been used successfully to rear first feeding larvae with small mouth gapes. The
smallest Acartia spp. nauplii used by Schipp et al. Ž1999. to rear Lutjanus johnii larvae
were 65 mm in width. Toledo et al. Ž1997. used Acartia sp. and Oithona sp. nauplii that
were G 50 mm wide and Pseudodiaptomus sp. nauplii that were G 80 mm wide to rear
the grouper Epinephelus coioides. Gut contents showed that these larvae readily
consumed the larger Pseudodiaptomus nauplii at first feeding. G. imparipes is a
temperate water species but could be used for rearing the larvae of some tropical fish
with appropriate temperature control of the copepod cultures.
In general, G. imparipes was very tolerant to different salinities, supporting the
findings of Rippingale and Hodgkin Ž1974.. Thus, this copepod species is suitable for
rearing fish species whose larvae occur in either estuary or oceanic environments. Also,
G. imparipes is able to survive rapid drops in salinity, which has proved effective in
 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262 259

removing contaminants from intensive copepod cultures ŽPayne and Rippingale, in

press.. Presumably, G. imparipes nauplii, like Artemia nauplii, could tolerate rinsing
with freshwater to reduce bacteria prior to feeding fish.
For copepods provided with excess food, differences in salinity had little effect on
maturation and nauplius production. For animals that were starved, significant differ-
ences in survival did occur. This has also been observed with other estuarine calanoid
copepods ŽRippingale and Hodgkin, 1977; Rippingale and Crossland, 1993. and sug-
gests that osmoregulation in these animals requires energy, which has been confirmed in
Eurytemora affinis ŽGonzalez and Bradley, 1994.. Thus, in the present study, energetic
cost of osmoregulation to G. imparipes can only be demonstrated during starvation.
Significantly longer survival in starved copepods maintained at 18‰ and 27‰ ŽFig. 2.
indicates a reduced need for energy expenditure at these salinities. This is supported by
shorter maturation time at 27‰ ŽFig. 3.. While provision of excess food allowed
copepods to maintain high growth and fecundity at high and low salinities, any condition
that further increases stress in an intensive culture environment should be avoided.
Hence, cultures should be maintained at 18–27‰.
Time to maturity of 10.4 days at 27‰ compares well with previous work. Using the
same methods, Payne and Rippingale Ž2000a. recorded a maturation time of 10.1 days in
G. imparipes fed excess I. galbana at 27‰ and 258C. However, this previous study also
recorded nauplius production of 25.1 naupliirfemalerday under exactly the same
conditions compared to 17.1 in the present study. For both studies, nauplii were
collected from cultures started from copepods collected in 1995; these cultures were
maintained without further collections of copepods from the wild and on a monodiet of
I. galbana. Nauplii used by Payne and Rippingale Ž2000a. were collected from cultures
operating approximately 7 months prior to those used to supply nauplii for the present
study. Thus, reduced fecundity in the latter could be a result of inbreeding depression or
nutritional deficiencies associated with long-term use of a monodiet. Given the magni-
tude of the decrease, this requires further investigation.
At 88C, high survival of G. imparipes nauplii was recorded after 12 days. Thus,
nauplii can be collected daily from intensive culture, placed in storage at 88C and on the
12th day, a considerable number of accumulated nauplii would be available for feeding
to fish larvae. This may be particularly useful for larvae at the critical first feeding stage.
For this purpose, further studies are necessary to determine the effect of increased
nauplius stocking densities Žperhaps with aeration. on storage time. Survival of adult G.
imparipes stored at 88C was high after 42 days. During periods when copepod cultures
are not required, adult copepods can be stored at this temperature and every 42 days,
they could be removed from storage and used to produce the next generation. This
cohort could be grown to maturity and then stored at 88C for a further 42 days or kept as
nauplii in cool conditions. Alternatively, removed adults could be well fed for 12–24 h
at room temperature and returned to storage. Thus, copepods can be kept for prolonged
periods with minimum maintenance.
Neither refrigerator maintained a particularly stable temperature. Hence, poor survival
of copepods stored at 48C was probably a result of temperatures falling to 2.58C in this
refrigerator. Presumably, copepod enzyme activity is impaired at this low temperature.
Alternatively, overnight temperatures approaching freezing may have occurred in this
260 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262

refrigerator causing damage to copepod body tissues. Poor temperature control in this
study prevented accurate determination of the minimum storage temperature for G.
imparipes nauplii. However, these results do have relevance to the many commercial
aquaculture facilities that make use of domestic refrigerators for live food storage.
Støttrup et al. Ž1999. hatched nauplii from eggs of the calanoid copepod A. tonsa stored
at 48C for 12 weeks. However, these workers were interested in the effect of storage on
copepod lipid content and did not record viability.
Fatty acid content of G. imparipes nauplii reflects that of their algal diet ŽPayne et
al., 1998.. I. galbana is high in DHA and very low in EPA and AA, while N. oculata
contains EPA and AA and no DHA ŽDunstan et al., 1993.. Hence, nauplii enriched with
I. galbana in the present study recorded an increased content of DHA but not of EPA or
AA. Quantitative increase of total fatty acids, including DHA, after 6-h enrichment
suggests tissue storage of fatty acids by nauplii. When N. oculata was added to the
enrichment diet, EPA and AA contents increased along with total fatty acid content.
Payne and Rippingale Ž2000a. attributed poor growth and survival of G. imparipes
nauplii fed a monodiet of N. oculata to either poor ability of nauplii to capture the small
cells of this alga from the water column or poor digestion of the hard cell wall. The
present study suggests that nauplii can ingest N. oculata cells. However, it is not clear
whether increased EPA and AA levels in nauplii are a result of undigested N. oculata
cells in the gut or of tissue accumulation of fatty acids from digested cells.
Little data is available on the quantitative fatty acid content Žexpressed in terms of
dry body weight. of cultured copepod nauplii. Norsker and Støttrup Ž1994. recorded
0.8–1.17 ng DHArindividual in harpacticoid nauplii fed various diets. Calculations
from data in Table 1 indicate G. imparipes nauplii contained a similar level: 0.75–1.11
ng DHArindividual. In a recent review, Sargent et al. Ž1999. stated that the ideal larval
food Žbased on the lipid composition of fish eggs. should contain approximately 17.4,
9.5 and 1.2 mgrgDW of DHA, EPA and AA, respectively. This is somewhat higher
than measured for early G. imparipes nauplii. However, total fatty acid content rapidly
increases in G. imparipes as they grow; from 59.5 mgrgDW in early nauplii Žpresent
study. to 438 mgrgDW in adults ŽPayne et al., 2001.. This increase occurs in other
copepod species ŽNorsker and Støttrup, 1994; Evjemo and Olsen, 1997. and indicates
that any increase in copepod size will result in a rapid increase in their value as food for
fish.
The ideal larval diet proposed by Sargent et al. Ž1999. contains a DHA:EPA and
EPA:AA ratio of 1.8 and 8, respectively. These ideal ratios were not recorded in G.
imparipes nauplii. Clearly, prolonged enrichment with I. galbana alone increases DHA
in nauplius tissues but does not provide desirable ratios. These ratios improved greatly
with the addition of N. oculata during enrichment. Further improvement may be
achieved by increasing the proportion of this alga relative to I. galbana, thereby
increasing EPA and AA relative to DHA. Alternatively, other algal species, such as
Rhodomonas salina or PaÕloÕa lutheri, that may already contain close to desirable fatty
acid ratios ŽBrown et al., 1997. could be used.
This study enables further refinement of intensive culture techniques for G. impar-
ipes. It provides a method for increasing the number of nauplii available for feeding to
larval fish at a particular time and methods for improving the nutritional suitability of
 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262 261

nauplii for fish larvae. This will increase the cost effectiveness of using G. imparipes as
live food in commercial fish hatcheries.

Acknowledgements

Financial support was received from the Fisheries Research and Development
ŽFRDC; Project No. 96r398.. The assistance of Dr. R. Longmore of the School of
Pharmacy, Curtin University, in fatty acid analyses is gratefully acknowledged.

References

Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, G.A., 1997. Nutritional properties of microalgae for
mariculture. Aquaculture 151, 315–331.
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.
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.
Evjemo, J.O., Olsen, Y., 1997. Lipid and fatty acid content in cultivated live feed organisms compared to
marine copepods. Hydrobiology 358, 159–162.
Gonzalez, C.R.M., Bradley, B.P., 1994. Salinity stress proteins in Eurytemora affinis. Hydrobiology 292r293,
461–468.
Kraul, S., Nelson, A., Brittain, K., Ako, H., Ogasawara, A., 1992. Evaluation of live feeds for larval and
postlarval Mahimahi Coryphaena hippurus. J. World Aquacult. Soc. 23, 299–307.
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.
Ohno, A., Okamura, Y., 1988. Propagation of the calanoid copepod, Acartia tsuensis, in outdoor tanks.
Aquaculture 70, 39–51.
Payne, M.F., Rippingale, R.J., 2000a. Evaluation of diets for culture of the calanoid copepod Gladioferens
imparipes. Aquaculture 187, 85–96.
Payne, M.F., Rippingale, R.J., 2000b. Rearing West Australian seahorse, Hippocampus subelongatus,
juveniles on copepod nauplii and enriched Artemia. Aquaculture 188, 353–361.
Payne, M.F., Rippingale, R.J., in press. Intensive cultivation of the calanoid copepod Gladioferens imparipes.
Aquaculture.
Payne, M.F., Rippingale, R.J., Longmore, R.B., 1998. Growth and survival of juvenile pipefish Ž Stigmatopora
argus . fed live copepods with high and low HUFA content. Aquaculture 167, 237–245.
Payne, M.F., Rippingale, R.J., Cleary, J.J., 2001. Cultured copepods as food for West Australian dhufish
Ž Glaucosoma hebraicum. and pink snapper Ž Pagrus auratus . larvae. Aquaculture 194, 137–150.
Rippingale, R.J., Crossland, P.A.M., 1993. Food availability and salinity tolerance in the copepod Eurytemora
affinis Poppe. Arch. Hydrobiol. 75, 357–362.
Rippingale, R.J., Hodgkin, E.P., 1974. Predation effects on the distribution of a copepod. Aust. J. Mar.
Freshwater Res. 25, 81–91.
Rippingale, R.J., Hodgkin, E.P., 1977. Food availability and salinity tolerance in a brackish water copepod.
Aust. J. Mar. Freshwater Res. 28, 1–7.
Sargent, J., McEvoy, L., Estevez, A., Bell, M., Henderson, J., Tocher, D., 1999. Lipid nutrition of marine fish
during early development: current status and future directions. Aquaculture 179, 217–229.
Schipp, G.R., Bosmans, J.M.P., Marshall, A.J., 1999. A method for hatchery culture of tropical calanoid
copepods, Acartia spp. Aquaculture 174, 81–88.
262 M.F. Payne, R.J. Rippingaler Aquaculture 201 (2001) 251–262

Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R., Sargent, J.R., 1999. Natural copepods are
superior to enriched Artemia nauplii as feed for halibut larvae Ž Hippoglossus hippoglossus. in terms of
survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr. 129,
1186–1194.
Støttrup, J.G., Norsker, N.H., 1997. Production and use of copepods in marine fish larviculture. Aquaculture
155, 231–248.
Støttrup, J.G., Bell, J.G., Sargent, J.R., 1999. The fate of lipids during development and cold-storage of eggs
in the laboratory-reared calanoid copepod, Acartia tonsa Dana, and in response to different algal diets.
Aquaculture 176, 257–269.
Sun, B., Fleeger, J.W., 1995. Sustained mass culture of Amphiascoides atopus a marine harpacticoid copepod
in a recirculating system. Aquaculture 136, 313–321.
Toledo, J.D., Golez, S.N., Doi, M., Ohno, A., 1997. Food selection of early grouper, Epinephelus coioides,
larvae reared by the semi-intensive method. Suisan Zoshoku 45, 327–337.
Toledo, J.D., Golez, M.S., Doi, M., Ohno, A., 1999. Use of copepod nauplii during early feeding stage of
grouper Epinephelus coioides. Fish. Sci. 65, 390–397.
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.