Aquacultural Engineering 1 (1982) 45-53

AUTOMATION IN STOCK-CULTURE MAINTENANCE AND

JUVENILE SEPARATION OF THE MYSID
M YSIDOPSIS BAHIA (MOLENOCK)

PHILIPPE LINGERt and PATRICK SORGELOOS~:

Artemia Reference Center, State University of Ghent, J. Plateaustraat 22, B-9000 Ghent, Belgium

ABSTRACT

A batch system with subgravelfilterfor stock-culture maintenance o f Mysidopsis bahia

in artificial sea water is described. The feeding o f mysids on Artemia is optimized
through the cyclic pumping o f brine shrimp instar I nauplii from a refrigerator to the
stock-cultures. An easily constructed incubator-separator apparatus is described for
the standardized harvesting o f mysid ]uveniles to be used as test organisms.

INTRODUCTION

The estuarine mysid shrimp Mysidopsis bahia Molenock (1969) is regularly being used
as a suitable test organism in toxicological (Bahner et al., 1977; Nimmo et al., 1977;
Anonymous, 1978) and nutritional studies (Johns et al., 1981). Therefore Mysidopsis
bahia juveniles were selected as test organisms in standardized predator-prey experi-
ments to evaluate the nutritional value of various Artemia products, e.g. decapsulated
cysts and nauplii from different geographical origin, various preparations of fed and
unfed nauptii, etc.
According to the literature stock cultures of this mysid should be maintained in
open flow-through systems with natural sea water (Clutter and Theilacker, 1971;
Anonymous, 1978). An original technique was worked out for stock maintenance in
batch systems with artificial sea water in situations where no running sea water is
available. Food distribution to the stock cultures was automated and an incubator-
separator system developed for the standardized harvesting of mysid juveniles to be
used as test organisms in the predator-prey experiments.
t Research assistant and ~ research associate at the National Fund for Scientific Research (Belgium).
45
Aquacultural Engineering 0144-8609[82[0001-0045]$02.75 © Applied Science Publishers Ltd,
England, 1982
Printed in Great Britain
46 P. LINGER, P. SORGELOOS

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Fig. 1. Schematic diagram of the aquarium set-up for stock-culture maintenance of Mysidopsis
bahia with details on the subgravel filter system. (1) Inflow of food, (2) air-water lift, (3) sub-
gravel filter system, (4) marine sand, (5) crushed seashells, (6) silex grains, (7) perforated plastic
plate.

STOCK-CULTURE MAINTENANCE

Glass aquaria (60 × 30 × 35 cm; capacity 60 litres) were equipped with a subgravel
filter as schematically outlined in Fig. 1. A perforated plastic plate was installed at the
bottom of each aquarium and covered with three filtering layers: i.e. silex-grains at the
bottom (average particle size 3 ram), fine marine sand at the top, and a thin layer of
crushed seashells in between. Two air-water lifts per aquarium circulated the water
through the subgravel filter at a flow rate of 50-80 litres rain-1 m -2 filter bed, assuring
optimal bacterial activity in the filter (Kinne, 1976).
Artificial sea water prepared according to the formula of Dietrich and Kalle (Kalle,
1971) but enriched with NaHCO3 until the pH stabilized in the range 8.0-8.5 (Spotte,
1979), was selected as a suitable medium. The salinity of the 0.2 gm filtered sea water
 AUTOMATIONOF MYSID STOCK-CULTUREMAINTENANCE 47

was adjusted to 30 ppt and the temperature maintained at 23°C -+ 2°C (Johns, personal
communication).
Since mysids prefer dimmed light conditions (Kinne, 1977) the top and front side
of the aquaria were covered in such a way as to allow only diffuse indirect illumina-
tion at 10-50 lux at the back side of the aquaria. The fluorescent light tubes in the
culture room were set at a 12 h light, 12 h dark photoperiod.
In view of their high food value for Mysidopsis bahia (Johns etal., 1981)and their
low content of chlorinated hydrocarbons (Olney et al., 1980), Artemia nauplii from
the Macau-Brazil variety were selected as food source. One food distribution per day
as advised in the literature (Clutter and Theilacker, 1971) resulted in an inefficient
food uptake, i.e. satiated juveniles and adult mysids continued to chase brine shrimp
nauplii and bite them to pieces without consuming them. Whereas these decaying
Artemia fragments were continuously washed out in open flow-through systems, they
overloaded the subgravel filter in batch systems. Maximal food uptake by the mysids
with minimal pollution effects was assured by four distributions per day of about
40000 Artemia nauplii to each aquarium (see below for technical details on auto-
matic food-distribution).
The mysid stock cultures have been successfully maintained in batch conditions for
over one year. Each stock-aquarium continuously held a few hundred gravid females.
Aside from the work concerning food preparation and distribution (see below) the
following operations were performed routinely:
1. Daily from Monday to Friday the filter bed was raked and quick checks were
made on water temperature, air-water lift activity, food presence, and overall
status of the population (abnormal mortality, behaviour of adults);
2. Although in previous experiments the water quality was never a problem, the
dissolved oxygen content, salinity, ammonia-N, nitrite and nitrate level were
monitored once a week. As soon as the nitrate level exceeded 20 ppm, 50% of
the culture water was changed;
3. Every month the reproductive activity of the population was checked, i.e. a few
spawnings were controlled for the size of the brood, the viability, and malforma-
tions of the juveniles. As a function of age, a healthy female was carrying from 5
to 20 young;
4. As advised by Kinne (1976), the filter bed was renewed at least once a year.

AUTOMATION IN F E E D I N G O P E R A T I O N S

In order to minimize the manual work of feeding the three 60 litre culture-stocks with
Artemia nauplii, the Artemia cyst incubation and nauplii distribution was automated
through the operation of a microprocessor universal timer (TMS 1122, PBJ Electronik,
Denmark). Manual intervention was required only three times a week for about 15 min.
48 P. LEGER, P. SORGELOOS

C2]:[l - -- 3 (31D" :

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Fig. 2. Set-up for the automatic incubation and hatching of Artemia cysts. A, plastic bag with
dry cysts; B, plastic bag with incubated cysts; C, sea water reservoir; (1) continuous aeration;
(2) fluorescent light tube; (3) aeration from air-pump activated by timer.

The set up for nauplii production is schematically outlined in Fig. 2. Three times a
week 25 g Macau cysts were hatched in 10 litres of sea water at 30 ppt salinity, 23°C,
and under continuous illumination (Sorgeloos, 1980). In order to assure a maximum
hatching efficiency as well as a homogeneous harvest of instar I nauplii, the cysts were
hydrated and incubated exactly 30 h prior to harvesting, i.e. at the preset moment,
air-pump (3) was switched on by the timer and sea water was pumped from reservoir
(C) into the hatching bag containing the dry cysts.
Manual harvesting of the Artemia nauplii was done as follows: the aeration was
switched off and a black plastic cylinder was fitted over the hatching bag, leaving the
lower part of the funnel illuminated. Due to their low specific gravity, the empty cyst
shells floated at the water surface while the phototactic nauplii of Macau-origin con-
centrated in the funnel of the hatching bag. After 10 rain separation the nauplii were
siphoned off on a 110/am filter screen, rinsed with chilled sea water (30 ppt, 2-5°C)
and diluted to a final concentration of about 4000 nauplii m1-1 into the food stock
container (1 litre capacity, see Fig. 3) which was installed in a refrigerator. Automation
in food distribution was greatly facilitated by the possibility of storing live instar I
Artemia in the refrigerator, i.e. provided the nauplii were continuously kept in suspen-
sion by slight aeration, over 95% survival with minimal losses of their individual dry
weight was obtained after 48 h storage of Macau-nauplii at 2-5°C (IAger et al., 1981 a).
Four times a day the timer activated three easily assembled peristaltic pumps (see
Fig. 4) assuring the distribution of nauplii from the stock-cylinder in the refrigerator
to the three stock-aquaria. A few seconds before the pumping stopped, the feed lines
were automatically rinsed with sea water, i.e. the electromagnetic valve (1) opened and
sea water was drained into the food distribution tube; as a result of the hydrostatic
 AUTOMATION OF MYSID STOCK-CULTURE MAINTENANCE 49

(2)

(3)

Fig. 3. Schematic diagram of the food stock-container with accessories. (1) Electromagnetic
valve activated by timer, (2) inflow of sea water from rinsing reservoir, (3) feed line connected
with inflow tube o f peristaltic pumps, (4) one-way valve.

pressure of the sea water inflow, the one-way valve (4) closed off the supply of nauplii,
and clean sea water was pumped through the feed lines into the stock-aquaria.
The present automations in food preparation and distribution not only greatly
facilitated but at the same time optimized mysid stock-culture maintenance. Manual
interventions on Mondays, Wednesdays, and Fridays were limited to the following
operations:
1. interruption of the aeration in hatching bag A (or B)
2. haivesting of the nauplii from hatching bag A (or B)
3. cleaning of the food stock-container in the refrigerator
4. transfer of the nauplii into the food stock-container
5. rinsing of hatching bag A (or B)
6. restoring the aeration in hatching bag A (or B)
7. input of 25 g cysts in hatching bag B (or A)
8. filling of the sea water reservoir with 10 litres of 30 ppt sea water
9. transfer of the tube for sea water inflow to hatching bag B (or A)
50 P. LEGER, P. SORGELOOS

(1)

a-b
Fig. 4. Schematic diagram of peristaltic pumps for Artemia nauplii distribution. (1) Inflow (3 mm
inner diameter) connected with food stock-container, (2) silicone tubing (6 mm inner diameter)
fixed on two supports, (3) rotating PVC-cam, (4) outflow (1 mm inner diameter) to stock-aquarium,
(5) Crouzet synchronous motor-reductor 20 rpm, (6) electric activation by timer.

INCUBATOR-SEPARATOR FOR THE STANDARDIZED HARVESTING OF

MYSID JUVENILES

For comparative nutritional and toxicological tests with mysid juveniles, it is always
imperative to use experimental animals of a specific age. The continuous culture
system for Artemia, Daphnia, and other invertebrates as developed by Sorgeloos and
Persoone (1973) was modified for short-term incubation of gravid mysids, and auto-
matic separation of their 0-24 h old offspring. Advantage was taken of two behavioural
characteristics of mysid shrimps in the development of the present incubator-separator
system:
an increase of the water-temperature by 2°C stimulates the females to release
their offspring (Johns, personal communication);
- upon birth juvenile mysids do not swim but sink to the b o t t o m or are carried
away by currents. A few minutes later they moult into a swimming stage.
The apparatus (Fig. 5) was composed of two interconnected cylindroconical vessels,
which were entirely made of acrylic glass and through which water circulated by the
force of an air-water lift. Vessel A (25 litre capacity) was equipped with a filter cylinder
 AUTOMATIONOFMYSIDSTOCK-CULTUREMAINTENANCE 51

~2J

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Fig. 5. Schematic diagram of incubator-separator apparatus for juvenile harvesting inMysidopsis
bahia. A, incubator for adult mysids with filter basket (1), aeration line (2), thermostat-heater (3),
and stopcock (5) with large opening; B, separator box for mysid juveniles with filter screen (4),
watertight fixing system (a) of lid and stopcocks (6 and 7).

(1200/~m screen) which retained adult mysids but let juvenile mysids and brine shrimp
nauplii pass. Water in vessel A was aerated with an airstone and kept at 25°C with a
submersible heater with a thermostat. Stopcock (5) at the end of the funnel had a
wide opening (10 mm inner diameter) to facilitate passage of the mysid juveniles.
52 P. LEGER, P. SORGELOOS

Vessel B (4 litre capacity) was made up of a cylindroconical bottom part on to

which a watertight cover was fixed with rubber bands and spring-back binders. The
bottom part held a 500/am filter screen, which for about half its surface was covered
by silicone strips that provided shelters for the mysid juveniles and at the same time
assured a more efficient suction of the Artemia nauplii through the filter. The cover
of vessel B was equipped with a purge tap (6) and with an open tube to be connected
to stopcock (5). The funnel was connected to the lateral air-water lift which pumped
water and Artemia nauplii from B into A.
A homogenous dispersion of the Artemia nauplii over the entire apparatus was
furthermore achieved by shading vessel A, i.e. the phototactic Artemia nauplii (from
Macau origin) migrated to the unshaded bottom part from where they were easily
circulated through B and back into A.
A few hours prior to routine operation, the incubator-separator was filled with arti-
ficial sea water (formula of Dietrich and Kalle, 30 ppt salinity, 0-2/lm filtered) and the
heating and aeration were switched on. In order to attain a food density of about one
Artemia nauplius per ml of culture medium about 30 000 freshly hatched nauplii were
added to vessel A. As soon as the temperature had stabilized at 25°C -+ 2°C (warmer
than in the stock aquaria), as many gravid females as the number of juveniles wanted
were collected from the stock cultures and transferred into the filter basket of vessel A.
After 24 h incubation a first batch of mysid juveniles could be harvested: i.e.
stopcock (5) and the air were closed off while stopcock (6) was opened. Upon discon-
necting the air-water lift tubing located just after stopcock (7), water could be drained
off from B. As soon as the cover of vessel B was empty, stopcock (7) was closed, vessel
B opened, and the mysid juveniles were collected.
If more juveniles were needed, vessel B was reassembled and connected to A, and a
new batch of freshly hatched Artemia nauplii was added. The second separation
always resulted in the greatest number of juveniles. More separations could be per-
formed but the number of juveniles was sharply decreasing from the third separation
onwards.
As soon as the separation was finished the adult mysids were collected from vessel
A and put back into the stock-aquaria. The entire apparatus was rinsed with tap water
and air dried.
This incubator-separator system has been successfully used for routine production
of hundreds of mysid juveniles at a time. The high reproducibility of survival and
growth results obtained with these juveniles in standard culture tests (IAger et al.,
1981b) were the best proof of the suitability of this new incubator-separator system
for the production of experimental test-organisms.

ACKNOWLEDGEMENTS

We are very indebted to Allan D. Beck and D. Michael Johns from the US-EPA
Environmental Research Laboratory at Narragansett (RI-USA) for providing us with a
 AUTOMATION OF MYSID STOCK-CULTURE MAINTENANCE 53

stock-culture of Mysidopsis bahia and for their information with regard to mysid
culturing; to Paul Vanhaecke for the transport of the mysids to our laboratory; to
Freddy Detry and Johan Tavernier for their suggestions and technical help in develop-
ing the various systems, and to Edmonde Jaspers for proofreading the manuscript.

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