Aquaallture

Aquaculture 136 (1995) 313-321

Sustained mass culture of Amphiascoides atopus a

marine harpacticoid copepod in a recirculating
system
Bin Sun ‘, John W. Fleeger *
Department of Zoology and Physiologym Louisiana State University, Baton Rouge, LA 70803, USA

Accepted22 May 1995

Abstract

A sustainable mass-culture system (4 mz basal surface area) of the meiobenthic harpacticoid

copepod Amphiascoides atopus, capable of producing in excess of one million individuals and over
5 g dry weight biomass per day, was successfully designed and implemented. The system consists of
culture tanks containing a shallow layer of limestone cobble facilitating naupliar and juvenile cope-
podite development. Adults and older copepodites swim into the overlying water which is recirculated
and filtered, expediting their clean capture. A. atopus ranges in length from 0.19 mm (for Nauplius
I) to 0.92 mm in the adult, and up to 5 pg dry weight. Copepods were fed either cultured algae
(Chaetocerous muelleri, strain Chaet 10) or commercial fish-flake food, and grew well on each diet
or a mixture. Amphiascoides atopus life history patterns are favorable for mass culture. Clutch size
averages 24 eggs per ovigerous female, and new egg clutches are produced every 34 days. When
cultured at 23 “C, life expectancy is about 3 mo, and generation time is less than one month. Preliminary
laboratory feeding experiments with various shrimp and fishes and suggest that A. atopus may prove
to be an excellent food source for larvae, post-larvae and juveniles and may serve as an alternative
live prey to brine shrimp in mariculture applications.Key words: harpacticoid copepod culture, meiob-
enthos, fish and shellfish mariculture.

Keywords: Harpacticoid copepod culture; Meiobenthos; Fish and shellfish maricultnre

1. Introduction

The need for improved living food sources for developing larvae, post-larvae and juvenile
fish and crustaceans is an important problem facing marine aquaculturists. The rotifer

* Corresponding author.
’ Louisiana Department of Natural Resources, Coastal Restoration Division, P.O. Box 94396, Baton Rouge,
LA 70803, USA.

0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDIOO44-8486(95)01064-5
314 B. Sun, J. W. Fleeger/Aquaculture 136 (1995) 313-321

Brachionus plicatilis and the branchiopod crustacean Artemia have been commonly used
as prey during these early but critical periods in life history, but do not always promote
optimal growth. Problems associated with rotifers and brine shrimp include nutritional
deficiencies and inappropriate prey size (Kahan, 198 1; Scott and Middleton, 1979; Howell,
1979; Kahan, 1980; Leger et al., 1986). Alternative food sources that overcome these
inadequacies and promote adequate growth are needed.
Several lines of evidence suggest that harpacticoid copepods may serve as exceptional
live prey for hatchery-reared fishes and crustaceans (Kahan, 1980; Watanabe et al., 1983).
Harpacticoid copepods are infaunal or epibenthic crustaceans that sometimes enter near-
bottom waters and frequently serve as essential or obligate prey for many larval, post-larval
and juvenile fishes (especially sciaenids, salmonids and flatfish) and crustaceans (Gee,
1989; Coull, 1990; McCall, 1992; McCall and Fleeger, 1995). Harpacticoids have a rela-
tively high caloric content per unit weight and superior nutritional value compared to many
traditional food sources (Kahan, 1981; Kahan et al., 1982; Volk et al., 1984; Chandler,
1986; Gee, 1989). Specifically, copepods tend to be rich in essential fatty acids, most
notably 22:6w-3 and 20:5wn-3, that are deficient in some strains of Artemia (Leger et al.,
1986; Norsker and Stottrup, 1994). Additionally, harpacticoids, from first nauplius to adult,
provide a broad spectrum of prey sizes (80 to > 900 pm in length and up to 3-5 pg dry
weight) suitable for ingestion by an equally broad size range of developing fish with small
gapes (Gee, 1989). Artemia may be too large to be suitable prey for many fish larvae or
post-larvae with especially small gapes (Kahan, 198 1). Many species of harpacticoids have
been propagated (Chandler, 1986), but no large-scale and high-yield sustained culture
system, with the exception of Tigriopus japonicus, see Fukusho, 1980; Fukusho et al., 1980,
suitable for mariculture application has been devised especially for substrate associated
species. Copepod species with desirable traits for mass culture should possess the following
demographic characteristics: high reproductive potential, short turnover time (from egg to
egg), and fast individual and population growth rates. Other important properties include a
diet flexible enough to allow growth on a variety of food sources and tolerance of a wide
range of physical environmental factors such as temperature and salinity.
A previously undescribed meiobenthic harpacticoid copepod species fouling fish and
mysid culture tanks in a commercial bioassy laboratory was brought to our attention. The
species, Amphiascoides atopus Lotufo and Fleeger, proved to be an excellent candidate for
mass culture. Amphiascoides has been frequently mentioned as a genus that may prove to
be ideal in fish mariculture due to its ease of culture and high growth rates under a variety
of temperature and salinity regimes (Walker, 1979; Ingole, 1994; Lotufo and Fleeger,
1995). The purpose of the study was to design a harpacticoid copepod culture system using
A. atopus that could sustain high output with low maintenance that might provide yields
suitable for large-scale mariculture applications.

2. Materials and methods

To determine if Amphiascoides atopus exhibits favorable demographic traits, single egg

clutches (average of 24 eggs) were placed in eight plastic square dishes (60 ml) and held
at 24 (range from 23-26) “C and 32 (range from 30-34) %Oartificial sea water. As soon
 B. Sun, J. W, FIeeger/Aquaculture 136 (1995) 313-321 315

as the eggs hatched, Chaetocerous muelleri, strain Chaet 10 (a planktonic alga that was
concentrated and refrigerated from bulk culture provided by the Department of Civil Engi-
neering at Louisiana State University) was added to chambers at about 50 million cells per
ml every other day. One third of the water in the dish was also replaced every other day just
before feeding. Observations were made every day for the entire life span of the hatched
nauplii (egg to egg). Characteristics such as average clutch size and survival were also
estimated from batch culture.
In order to determine the relationship between length and dry weight in A. atopus, eleven
size classes of juveniles and adults from 0.4 to 0.9 mm were chosen, and 20 to 50 individuals
were measured for each group dependent on the size of the group (more individuals were
used in the smaller size classes) under a stereo-microscope. Body length was defined as the
distance from the tip of the cephalsome (excluding the rostrum) to last body segment
(excluding the caudal rami). All individuals of each group were placed in a pre-weighed
aluminum pan and dried in an oven at 95 “C for 24 h after length measurement. Total dry
weight was measured with a Cahn Electrobalance (to 0.1 pg accuracy, Cahn Instrument
Company), and average dry weight per individual for each size class calculated.

2.1. Culture system

A prototype culture system (System I) was constructed first. The pump-driven recircu-
lating system consisted of five components: a centrifugal pump, culture tanks, collectors,
filters, and a reservoir tank (Fig. 1) . The water pump was a l/10 hp commercially-available
centrifugal pump used to recirculate water among culture tanks. The culture tanks were
three 40 1 glass aquaria (the bottom surface area was 0.13 m2) and five 28 1 plastic storage
boxes (basal surface area 0.19 m2). The bottom of each culture chamber was covered with
limestone cobble (approximately 1 cm in diameter) up to 2 cm in depth. Collectors consisted
of a collecting bag made from 120 pm nylon mesh and placed on the return locations of
recirculated water from the culture. Filtration was accomplished with a large, coarse filter
and a small, fine filter. The coarse filter was a wooden box (60 X 55 X 40 cm) filled with
small pieces of limestone, and the small filter was a 5 pm commercially-available drinking

@GFi
Fig. 1. Diagrammatic representation of the main components of the Amphiascoides atopus culture system.
316 B. Sun, J. W. Fleeger/Aquaculture 136 (1995) 313-321

water filter. This fine filter also served as a collecting structure. The reservoir tank was a
120 X 55 X 40 cm wooden tank also covered with limestone cobble. The water inside the
reservoir tank was aerated during recirculation. The total basal surface area for culture
(including filter and reservoir tank) was about 2 m2 and the total water volume was about
500 1.
A larger culture system (System II) was also built and modified based on our experience
with System I. This system was fabricated to attempt to promote higher yields and provide
experience with large-scale culture. A. atopus removed from this system was used to
maintain and culture grass shrimp (Puluemonetespugio) . The design of the System II was
modified from that of the prototype, and consisted of four components (pumps, culture
tanks, bag collectors and fine filters) without a large, course filter. Water circulated at a
constant rate through six culture tanks (four- 120 X 60 X 70 cm and two- 120 X 60 X 30 cm
tanks), and was driven by two centrifugal l/8 hp pumps. Air lifts were added to each of
the culture tanks to increase gas exchange among the limestone cobble. The total basal
surface of System II was about 4 m2 and the total water volume was about 1440 1.

2.2. Maintenance and harvest

After introducing harpacticoids, both culture systems were maintained at 24 (range from
23-26) “C and 32 (range from 30-34) %Owith an illumination cycle of 12 h light and 12
h dark (maintained by florescence lighting). For System I, l-2 million A. atopus were
introduced in July 1993. Harpacticoids were initially fed Chuetocerous muelleri at > 0.5
million cells/ml daily. Unfortunately, algal production stopped in the middle of September.
We switched the from algae to commercial fish flakes (TetraMarin, made in Germany) at
a level of 15 g every other day. A. atopus production did not appear to be interrupted. Waste
build-up in the coarse filter was cleaned periodically by washing out detritus and fecal
pellets from among limestone cobble. For System II, about 4 million A. atopus were
introduced in March 1994. Harpacticoids were fed exclusively Chuetocerous muelleri ( > 1
million cells per ml every other day) for about a month. Then fish flakes (20 g every other
day) were added to the culture after algal availability slowed to about half its former level.
Harvesting was begun about a month after A. atopus was introduced into each system.
No attempt was made to maximize collections and harpacticoids (all stages) were never
returned to the culture. Harpacticoids were collected and removed every other day. On each
collection the collecting bags and filter cartridge (for System I only, System II had no fine
filter) were detahed from each system. Harpacticoids in the collecting bags and the filter
cartridge were removed by rinsing the mesh bag and the cartridge into a 1000 ml beaker.
After collection, all harpacticoids were split four to six times in an eight chambered sample
splitter (Jensen, 1982). After each split density was reduced by one fourth by retaining two
randomly selected chambers for the next aliquot. Two chambers of the final split were
preserved and later enumerated in order to estimate total number of A. atopus removed on
each collection. Because A. autopus nauplii are benthic, very few were collected even
though the fine filter in System I could retain them. For System II, a submersible pump was
added to increase collection efficiency and remove waste products near the middle of the
trial. The bottom water of one of the culture tanks was manually pumped to a sieve until
the number of harpacticoids collected reached approximately 3-4 million (judged by eye).
 B. Sun, J. W. Fleeger/Aquaculture 136 (199.5) 313-321 317

Routine measures of ,population density in the culture systems were not attempted. Daily
harvest was calculated based on the total estimated yield of each collection.

3. Results

Arnphiuscoides atopus, Family Diosaccidae, develops through larval (nauplius) and

juvenile (copepodite) stages before a final molt to the adult stage. Nauplii and younger
copepodites are benthic in lifestyle, living in and among the spaces associated with the
limestone cobble. Older juveniles and adults are epibenthic, frequently swimming into the
water but tending to attach to the bottom and sides of the culture chamber. The dimensions
of adult A. atopus were 0.92 mm (SD: 0.06; n = 30) in length and 0.17 (SD: 0.014; n = 30)
mm in width. The length of the smallest nauplius (Stage I) was 0.19 mm (0.068 mm in
width). Average adult dry weight (body length > 0.8 mm) was 5.1 pg (SD: 0.9 n = 56).
The relationship between dry weight and length was also examined and there was a general
linear relationship between dry weight and body length although the minimum length
measured used was 0.4 mm (Y = 8.7X - 2.8 r= 0.94, where Y = dry weight and X = size
class length, Fig. 2).
Population growth and reproductive rates of this species are very high. The average turn-
over time (generation time from egg to egg) was between 21 and 26 days (at 30% and 24
“C) . Young mature females were able to produce an additional egg clutch within 48 h after
hatching. A. autopus typically carries two egg sacs and the average brood size was 24 eggs
per ovigerous female. A. utopus can feed on variety of food resources including settled
phytoplankton, fish flakes and plant materials. Large quantities of what appear to be lipid
storage sites inside the adult body can be seen with a microscope. A. utopus is strongly
photonegative, a property that is very useful when extracting and manipulating individuals.
After harvest, A. utopus was separated from the detritus by placing the entire collection in

o-
0.20 0.40 0.60 0.80 1.00 1.20

Body length (mm)

Fig. 2. The relationship between dry weight ( pg ) and body length (mm) of Amphiascoides atopus (see text for
linear regression statistics),
318 B. Sun, J. W. Fleeger/Aquaculture I36 (1995) 313-321

g 500

s
2 400
0

g
9 300
e

2 200

0”

100

Fig. 3. Estimated daily yield (in thousands) of Amphiascoides atapus over the 57 days of culture in System I.

one end of a plastic trough filled with water under strong fiber-optic lighting, while blocking
the light with black plastic from the other end. Adults and older copepodites quickly swim
to the dark end of the trough, producing a very pure collection of harpacticoids.
High and sustained production of A. atopus was maintained in both culture systems. For
System I, based on 57 days of continuous collection and removal every other day, the total
yield was 8.7 million individuals, an average of 0.15 million per day (Fig. 3). Of those
collected, 32.5% were males, 52.2% females and 15.3% copepodites. For System II, the
harvest lasted for about 5 months (Fig. 4). Using the same collecting procedure as System

7- 1

Fig. 4. Estimated daily yield (in millions) of Amphiascoides atopus over the 17 weeks of culture in System II.
Note that beginning in July, a submersible pump was used to increase capture rate.
 B. Sun, J.W. Fleeger/Aquaculture 136(1995) 3I3-321 319

I, the total yield of System II was 36.3 million for the first 10 weeks and averaged 0.5
million per day. Then a more aggressive collection method was adopted with the use of a
submersible pump to improve capture efficiency, and daily harvest increased dramatically.
The yield was maintained at 24 million individuals per day to provide sufficient quantities
of A. atopus to maintain grass shrimp. The total yield for the next 7 weeks was 144 million,
an average of 2.8 million per day. This high level of production was maintained through
this time with no visible sign of population decline. No effort was made to maximize yield,
and many more individuals could probably have been collected.

4. Discussion

Although marine harpacticoids have been frequently propagated in the laboratory (Hicks
and Coull, 1983; Chandler, 1986; Tietjen, 1988), there has been no successful large-scale
mass culture of meiobenthic copepods in the United States. Fukusho ( 1980) reported that
2-3 kg (wet weight) of the planktonic Tigriopusjuponicus can be harvested daily in large
(200 m3), outdoor tanks in Japan. Meiobenthic species that have been cultured in quantities
with promise for mariculture applications have been grown without substrate (Tseng and
Hsu, 1984; Kahan, 1979; Kahan, 1980). It is difficult to compare our results with other
published works because the publications cited above usually report growth rates rather
than yield and culture densities are reported in numbers per volume. Nevertheless, our
results for Amphiascoides atopus suggest a potential for aquaculture application because
yields were on the order of millions of harpacticoids per day (and grams of biomass) for
an extended time in a relatively small (4 m2) basal surface area. Certainly A. atopus appears
to have many advantages (e.g. flexible nutritional requirements, fast growth, high repro-
ductive potential) as a species for mass culture, For exampled. autopus collections remained
high for several weeks after we began using a more aggressive collection technique with a
submersible pump in System II, suggesting that this species responds well to exploitation.
Substrate may contribute to the exceptional yield. The addition of algae or fish flakes to the
culture allows the limestone cobble to become coated with biofilms and bacteria, and the
very large total surface area available for growth, especially for the benthic larval and
juvenile stages, among the cobble appears to facilitate growth.
We did not measure population densities or growth rates in culture, nor did we attempt
to maximize harvests for A. atopus, so the full growth potential of this culture system is yet
to be determined. The daily yield in both culture systems fluctuated during the course of
experiment. Observed variations in daily capture were probably related the water volume
that was passed through the filters and collectors rather than population fluctuations within
the culture system. Smaller collections occurred on days when flow was decreased associated
with occasional blockage of filter collectors due to clogging by accumulation of detritus. In
addition, A. atopus appears to be tolerant of a wide range of temperate and salinity variations.
In preliminary experiments, temperatures below 10 “C slowed growth dramatically but most
individuals survived salinities from 1O-60%0 although best growth occurred from 25-35%0
(Sun, unpublished).
Amphiuscoides atopus may have important applications in mariculture. Copepods pro-
duced and removed from our larger culture system were fed to 100 grass shrimp every other
320 B. Sun, J. W. Fleeger /Aquaculture 136 (1995) 313-321

day, and the shrimp lived well and grew (Fleeger, unpublished). Preliminary evidence
suggests that grass shrimp can ingest a maximum of 150 A. afopus per hr, and that A. atopus
promotes growth at least as well as brine shrimp nauplii (Gregg, personal communication).
We have also found that A. atopus can be used to maintain darter gobies (Gobionellus
boleosoma, from 25-30 mm in length) and Pacific white shrimp (Penaeus vannamie from
7-25 mm in length) in the laboratory. Our observations suggest that adult A. atopus are
easily captured and ingested by all the shrimp and fish species we examined except for post-
larval (7-10 mm) red drum (Sciaenops ocellatus) ; adult A. atopus struggles and frees itself
before capture by red drum. Nauplii and younger copepodites were not offered to red drum
but may serve as more appropriate food to a wide range of larval fish. Thus, A. atopus is a
capable swimmer and may escape some predators. Tseng and Hsu ( 1984)) however, suggest
that fish larvae that have to swim and struggle to ingest prey grow stronger much faster than
do larvae fed on the more passive brine shrimp. Fatty acid profiles suggest that essential n-
3 fatty acids are well represented in A. atopus comprising some 28% of all phospholipids
(Carman, personal communication). Other harpacticoids have been found to have similar
values (Norsker and Stottrup, 1994)) further highlighting the potential for use in maricul-
ture. Other uses for large quantities of mass-cultured A. atopus are certainly possible, and
include but are not limited to genetic, physiological, toxicological and immunochemical
research.
Nevertheless, more research is needed before this harpacticoid species and our approach
to mass culture is proven to aid in mariculture. One need is to verify that the culture system
can be sustained for extended periods. Waste products, such as fecal pellets and decomposing
algae or fish flakes, accumulate among the cobble, and a low-maintenance method to clean
the culture system should be devised if a culture is to be maintained for many months to
years. Collection of nauplii is difficult in our current culture because they pass through the
mesh in our collecting filters and because nauplii do not emerge from the cobble as readily
as adults and copepodites. Perhaps a culture design such as Kahan et al. (1982) with a
floating mesh basket may provide nauplii directly to larval or juvenile fish. Growth rates of
juvenile fish fed A. atopus and brine shrimp separately need to be compared to determine
if A. atopus provides proper nutritional qualities and if it can be efficiently captured by
small predators. Certainly, the ability of other pelagic-feeding fishes to fed on A. atopus
needs to be investigated. Likely candidates are flatfish and tropical fishes, in addition to
fishes that are difficult to grow using Artemia as live prey.

Acknowledgements

We thank M. Macias and A. Todaro for their help in maintaining the culture systems and
Drs. R. Malone and K. Rusch for advice on system design and for providing algae. We also
thank Dr. B.C. Coull for commenting on an earlier draft of this manuscript. This copepod
was first shown to us by L. Johnson of Laboratory Technology Inc., New Orleans. We
greatly appreciate his continuing interest in this research project and we are especially
grateful for his allowing us continuing access to his facillities. Support from Sea Grant (R/
A-35PD) and LEQSF (RF/ 1993-96-RD-B-17) is appreciated.
 9. Sun, J.W. Fleeger/Aquaculture 136 (1995) 313-321 321

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