Aquaculture, 42 (1984) 217-224 217

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

ZOOPLANKTON INGESTION AND FEEDING BEHAVIOR OF

PENAEUS KERATHURUS LARVAE REARED IN THE LABORATORY

M. YOFERA, A. RODRIGUEZ and L.M. LUBIAN’

Institute de Investigaciones Pesqueras de Cbdiz (C.S.I.C.), Puerto pesquero s/n,
11006 Chdiz (Spain)
1Present address: Plan de Explotacibn Marisquera y de Cultivos Marinos de la Regi6n
SuratlPntica (PEMARES), Casa de1 Mar 5a Planta, Cddiz (Spain)
(Accepted 20 August 1984)

ABSTRACT

YCfera, M., Rodriguez, A. and LubiPn, L.M., 1984. Zooplankton ingestion and feeding
behavior of Penaeus kerathurus larvae reared in the laboratory. Aquaculture. 42: 217-
224.

Ingestion of Brachionus plicatilis and Artemia salina nauplii during larval and early
postlarval stages of Penaeus kerathurus has been investigated. Results show an active in-
gestion of rotifers from protozoea II until postlarva II. Artemia nauplii are eaten from the
mysis II stage. From the first postlarval stage, P. kerathurus requires a prey of greater size
than Brachionus. In the absence of such prey the animals tend to be cannibalistic.

INTRODUCTION

Feeding is one of the most important factors affecting larval development

of penaeids. Generally, the diets used during penaeid culture are based on a
mixture of algae, primarily diatoms, during the first stages. Brachionus plica-
tilis and Artemia salina nauplii are given from protozoea and later mysis or
postlarval stages, respectively (Hudinaga and Kittaka, 1967; Cook and
Murphy, 1969; Shigueno, 1975; Rodriguez, 1976; San Felic et al., 1976;
Lumare et al., 1978), although artificial diets have also been tested (Jones et
al., 1979). In these works the usefulness of diets is assessed by the survival
and growth of larvae. However, there is little information concerning inges-
tion rates and optimum concentration of food for penaeid larvae, although
the recent works of Emmerson (1980,1984) on P. indicus are worth noting.
In the present work we studied the ingestion of Brachionus and Artemia
nauplii in the early development stages of Penaeus keruthurus, a species of
some commercial interest in the Mediterranean region, and we attempt to
increase the understanding of food requirements and feeding behavior of
larvae of this prawn during culture.

0044~8486/84/$03.00 o 1984 Elsevier Science Publishers B.V.

218

MATERIAL AND METHODS

Rearing technique

Nauplii of Penaeus kerathurus Forskal were obtained from gravid females

collected in the spawning zone at the mouth of the Guadalquivir river (SW.
Spain) (Rodriguez, 1976). Three groups of nauplii (5000,lO 000 and 16 000
nauplii) from separate spawnings were placed in cylindrical fiberglass tanks
(500 1) filled with sea water at a temperature of 24°C and salinity of 36°/oo,
agitated by bubbling and an air-lift system, and subjected to continuous illu-
mination. The diatom Skeletonema costatum was supplied as food from pro-
tozoea I (PI), to maintain a concentration of 50-100 X lo3 cells ml-‘. Dur-
ing mysis (MI-MIII) the rotifer B. plicatilis was supplied in place of the dia-
toms. Freshly hatched Artemia nauplii were supplied when the prawn larvae
moulted to postlarva I (PLI). From this moment about one quarter of the
total water volume was renewed each day. With this rearing technique the
survival at stage PLIII in the three tanks was 87%, 72% and 89%, respective-
ly. The body length of larvae was measured according to the criterion de-
scribed by Lumare et al. (1978).

Ingestion measurements

Three organisms were tested as food for larvae: two strains of different
sizes of Brachionus plicatilis (strain Bs and strain S-l), and Artemia salina
nauplii (from Cidiz salt marshes). Size and dry weight of these organisms
are shown in Table I. The weight of Artemia nauplii and P. kerathurus larvae
was measured by the same procedure as described in Yufera et al. (1983) for
B. plicatilis.
Experiments were carried out in flasks with 200 ml of filtered sea water,
with gentle agitation by bubbling, 36°/oo salinity and 24 f 1°C. Each flask
contained 20-30 larvae. The larvae and postlarvae were taken from mass
culture tanks. Attempts were made to take individuals at the beginning of
each larval stage, but it was not always possible due to the rapid develop-
ment of the stages, and to the gradual loss of synchronism of development in
the later stages. For each larval stage, several flasks with different initial food

TABLE I

Size range (95% of individuals) and dry weight of the prey organisms supplied as food for
P. kerathurus larvae

Prey Size (pm) Weight (fig ind.-‘)

B. plicatilis, strain Bs 100-160 0.05

B. plicatilis, strain S-l 160-248 0.19
Artemia nauplii 430-530 2.43
 219

densities were examined for the food decrease over 24 h. Rotifer densities
were estimated at intervals of 1 to 3 h, in order to obtain a significant, but
not excessive, difference during each period. Survival data were taken at 24
h. To determine ingestion, only those periods were considered when the
rotifer density in larvae-free controls remained constant.
We assumed that the rate of food decrease (K) is constant in each experi-
ment, and it can be calculated as the slope of the regression line fitted to In
of food concentration against time. Thus, the ingestion rate (I) was calcu-
lated by the following equation:

I = (K/P) $!?zTn

where Co and Ct are the initial and final food concentrations in the period
considered, and P is the number of larvae per ml. The ingestion rate was
plotted against the mean food concentration (dm) of each period.
The ingestion of Artemia was determined by the same procedure, but in-
gestion data were taken over 24 h because the density of nauplii in larvae-
free controls was constant, and there were small differences between initial
and final food densities in the experiments.
Selection between the two Bruchionus strains was examined using the fol-
lowing index, derived from Ivlev (1955) cited by Yurochko (1976):

r-p
Electivity = -
r+p
where r = 5%of one organism in the total ingested; p = 5%occurrence of the
same organism in the medium. Thus, the electivity ranges between +l and
-1, and is a measure of positive or negative selectivity.

RESULTS

The range of body length and approximate duration of each development

stage are summarized in Table II. These length data fall between those re-
ported by Heldt (1938) for natural and cultured organisms (noting that post-
larva I corresponds with mysis IV in Heldt’s nomenclature), and they are
very similar to lengths cited by Lumare et al. (1978) for cultured P. keru-
thurus. An enhancing overlap between different stages can be observed as
development advances. During protozoea, mysis and earlier postlarval stages
the decrease rate (K) of rotifers was uniform in each experiment (correlation
coefficients of logarithmic regression range between 0.94 and 0.99). Except
for protozoea I, which showed no ingestion of rotifers, all larval stages
showed an increase in the ingestion rate with increasing rotifer density with-
in the range tested (Fig. 1). When we compared the slopes of ingestion rates
versus food concentration for different stages, the maximal values were
found from PI1 to MI and the later stages showed a gradual decrease (Table
III). In the experiments with rotifer + Artemia nauplii, the ingestion of roti-
220

fers during mysis II -111 did not differ from the results obtained with rotifers
alone. However, postlarvae did not ingest rotifers when Artemia was present.
The experiments with a mixture of both rotifer strains (Fig. 2) showed a
higher ingestion of Bs individuals by protozoea II than by protozoea III
which prefers S-l individuals (the ingestion of Bs and S-l observed for PI11
in Fig. 2 is similar, but the density of Bs is about twice that of S-l). This sug-
gests a possible foodsize selectivity. However, a contradiction is found in

TABLE II

Larval and postlarval length and age (at the end of each stage) at 24” C

Stage Length (mm) Age (days)

NI-VI 0.40-0.60 2
PI 0.85-1.20 4
PI1 1.40-1.75 6
PI11 2.10-2.50 8
MI 2.60-3.25 10
MI1 3.10-3.80 12
MI11 3.60-4.10 14
PLI 3.90-5.25 16-17
PLII 4.10-5.50 18-19
PLIII 5.00-6.20 22-23

PROTOZOEA II PROTOZOEA III 1 MYSIS I

.
.
.*
.
.’
r=0.971
: .i/
IO 20

0 s-1

3 MYSIS II-III
l ES
A S-l + Es
POSTLARVA I-II
5 U S-l (+ Artemia)
H
z 20 1 _I

FOOD DENSITY (rotifers ml-’ )

Fig. 1. Ingestion of rotifers by P. kerathurus larvae. Squares show the ingestion of rotifers
from a mixture of rotifers + Artemia nauplii.
 221

TABLE III

Daily ingestion (no. larva-’ d-* and pg larva-’ d-l) and feeding (fig dry matter ingested/
dry weight of larvae) rates of P kerathurus feeding on Brachionus and Artemia nauplii

Larval Dry wt. Slope of Ingestion rates Feeding rates

stage of rotifer
prawn ingestion (No. larva-’ d-l) (pg larva-’ d“)
larva rate
(UP)

Brachionus plicatilis
Food density (rot. ml-‘) 10 20 10 20 10 20
PI 2.2
PI1 9.9 1.10 253 518 48.3 98.5 4.88 9.97
PI11 22.0 0.86 263 470 50.1 89.4 2.28 4.06
MI 45.9 1.14 248 522 47.2 99.2 1.03 2.16
MII-III 72.6 0.48 146 261 27.8 49.7 0.30 0.68
PLI-II 95.6 0.35 128 213 24.3 40.5 0.25 0.42

Artemia nauplii
MII-III 72.6 56 137 1.88
PLI-III 101.6 87 212 2.09

30 -( 3’
PROTOZOEA II PROTOZOEA II

A A
‘;,
A
20- A EfBs)= + 0.14 - 2-
7 7
.sz A
: A l
,I,
E(S-1)s - 0.16
3 . 7 A
A
1O- ;1- ,_’
. .
: I~, 4
.
2 8 100% d 100%

Ri,Lk ,
2 +
d
" G ryn l-h! 9

f2 SO-
% PROTOZOEA III PROTOZQEA III A
s
A A
z
I~,
20- A
A
A E(BS)_ - 0.08

A ‘:
A
,c E(S-lj= + 0.11
10- ox
. i.
100% l ** 100%

rqLl-L, I

10 20 30 I 2 3

(rot1fers ml-’ 1 OJg In-1 )

FOOD DENSITY

Fig. 2. Ingestion of the strains Bs and S-l of B. plicatilis

from a mixture of the two rotifer
strains. E(Bs) and E(S-1) indicate the mean values of Iviev’s electivity index for each
strain.
222

one of the experiments with PII fed Bs alone (Fig. l), which showed lesser
ingestion than in experiments with S-l rotifers alone. On the other hand, in
terms of dry weight of uptake, S-l always presented the higher values.
The results obtained with Artemia nauplii are shown in Fig. 3. Mysis II-
III and postlarvae showed an increase of ingestion rates with increasing food
density up to 15-18 nauplii ml- I. Above this density the ingestion rates re-
mained constant between 48 and 62 nauplii ind.-’ d-l in mysis stages, and
between 77 and 100 nauplii ind.-’ d-l in postlarvae. When a mixture of
Artemia nauplii + Bruchionus was added, the ingestion of nauplii was similar
to that in experiments with Artemia alone.
il Artemla
MYSIS II-III
n Artemia (+ rotifrr)

0 Artemia
POSTLARVA I-III
l Artemia (+ rotlfer)

FOOD DENSITY (nauplii.ml

-1 )

Fig. 3. Ingestion of Artemia nauplii by P. kerathurus. Black symbols indicate the inges-
tion of Artemia from a mixture of rotifer + Artemia nauplii.

To obtain an estimation of daily dry mass ingested, the ingestion values in

the regression line for concentrations of 10 and 20 rotifers ml-‘, and the data
on maximal ingestion rates for Artemia nauplii were used (Table III). The
maximal value of dry mass of rotifers ingested was about 100 Erg larva-’ d-’
during PI1 to MI. The dry mass of Artemiu ingested increased from 137 1.18
larvae-’ d-’ during MII-III up to 212 pg larva-’ d-’ in postlarval stages. The
feeding rates (dry weight ingested/larval dry weight) decreased gradually
with increasing larval weight, from 10 times the larval dry weight for PI fed
on Brachionus at 20 rotifers ml- ‘, to 0.42 times in early postlarval stages.
The feeding rate increased when larvae were fed on Artemiu nauplii from the
MI1 stage.
Survival at 24 h is shown in Table IV. In protozoea stages survival showed
only slight variations between the different initial food densities tested, and
mortality was never above 10%. Similar results were found for mysis larvae,
with mortalities below 14%. In PLI-III, survival ranged between 80% and
100% (the lesser values correspond to postlarvae fed on rotifers), except in
the controls without food, which showed. a high mortality (survival 52%-
72%). In these later stages, the observation of dead individuals reveals the
existence of cannibalism.
 223

TABLE IV

Larval and postlarval survival in the different experiments. Co and Ct are the initial and
final food density (ind. ml-‘) in 24-h experiments. A = Artemia nauplii

Stage Food co ct %Survival Stage Food Co Ct %Survival

PI1 BS 17- 2 92 MII-III A 42- 37 100

Bs 12- 2 92 A 34 - 26 96
S-l 17- 1 92 A 25 - 17 100
S-l 12- 1 100 A 17- 9 100
Bs + S-l 23- 6 92 A 15- 12 96
starved - - 90 A 15- 10 100
PI11 S-l 27 - 0.5 100 A 12- 8 100
s-1 15 - 0.1 100 A B- 2 100
S-l 6-O 100 A 5-2 92
S-l 4-o 100 starved - - 88
s-1 1-o 96 PLI-III s-1 41- 15 80
Bs + S-l 30 - 0 100 S-l 30- 12 88
starved - - 92 S-l lo- 2 84
MI S-l 19- 2 100 S-l 8-l 80
S-l 6 - 0.3 100 S-l+A 16+25 - 16+11 88
S-l 5- 1 86 A 55- 40 88
S-l 1-o 88 A 45- 20 84
starved - - 86 A 35- 28 100
MII-III S-l 35- 1 100 A 24- 9 96
S-l 26- 7 100 A 22- 15 92
S-l 25 - 0.5 92 A 14- 7 92
S-l 9-o 100 A 12- 3 96
S-l+A 28+12 - 24+9 100 A 6-2 96
starved - - 52-72

DISCUSSION

The above results show that rotifers are successfully caught and ingested
by larvae from stage PII. Artemia nauplii are ingested in quantity from stage
MII, with maximal ingestion rates in the postlarval stages. In contrast to data
obtained with Artemia, we have not found any stabilization of ingestion
rates in relation to rotifer concentration in the larval stages. The decrease in
the ingestion rate of rotifers with increasing size of prawn larvae can be ex-
plained by decreased catch efficiency because of the small size of Brachio-
nus. It is also possible that, due to the lack of synchronism, some animals
started to moult and their feeding decreased during the time of the experi-
ment. From PLI, P. herathurus requires a prey larger than Brachionus. Thus,
in the absence of other food, the rotifers are eaten, but when Artemia nau-
plii are present the rotifers are left. The survival of larvae, even starved lar-
vae, at 24 h is very high though gradually less in the more advanced develop-
mental stages. From PLI the individuals show a higher mortality in starvation
conditions, but more due to cannibalism than to inanition. However, it is
interesting to note that survival data are greatly influenced by the pre-
experimental feeding conditions.
224

The results obtained in the present study are supported by other work
with penaeids. Shigueno (1975) reported that P. juponicus ingested 46 nau-
phi larva-’ d-r during MIII-PLII, and 85 nauplii larva-’ d-* during PLII-
PLIV. Gopalakrishnan (1976) found a maximal ingestion of 510-200
nauplii larva-’ d-’ during mysis stages of P. marginatus at a food density of
5 nauplii ml-‘. Emmerson (1984) reported a maximal ingestion rate of about
300 rotifers larva-’ d-’ at 12-14 rotifers ml-’ initial food density for P. indi-
cus during PII-MI, and 187 nauplii larva-’ d-’ at 9 nauplii ml-’ initial food
concentration in postlarval stages. The differences found among the differ-
ent reports depend largely on the dry weights of both food organisms and
larvae.
It is obvious that the larval rearing of penaeids requires the addition of
prey with a gradual size increase. Generally, Bruchionus and Artemia are
used because of their availability in large quantities. Copepods are also used
in some cases: these organisms provide a wide range of prey size, from nau-
plius to adult, and their presence in the rearing tanks (due to the addition of
a cultured strain or to an uncontrolled natural population) probably affects
the final survival obtained in culture.

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