USE OF BENEFICIAL BACTERIA IN THE LARVAL
REARING OF THE SHRIMP Penaeus schmitti.
Elvira Alfonso1, 2, Elpido Beltrame 2, Edemar
R. Andreatta 2, Alitiene Lemos 2 y Jair Quaresma 2.
1. Centro de Investigaciones Marinas, Universidad
de La Habana, Cuba.
2. Laboratório de Camarões Marinhos, Centro de Ciências
Agrárias, Universidade Federal de Santa Catarina, Brasil.
This treatise was
accepted for publication in August 1996 in the Magazine of Marine Investigations
(published jointly by the University of Havana, Cuba and the University
Nacional Atóónoma of Mexico).
Translated from Spanish
to English with the use of Babelfish from AltaVista and WordReference.com
by Valerie Anne Edwards
Recently, some live microorganisms have appeared
on the market designed as waste degraders for Aquaculture. The effect of
two products on the larval rearing of the white shrimp Penaeus schmitti
was tested. This species is commercially cultured in Brazil and Cuba. Dosages
were evaluated, the products' role in water quality and the possible control
of the gut epithelium scaling syndrome of the animals, known as "Bolitas",
were studied. In order to analyze the results, the following have been considered:
survival, metamorphosis rate, larval quality and size of the postlarva1.
It was concluded that the tested products can be used even with the Protozoea
I stage. This has enabled the daily water exchange rate to be reduced from
100% to 30% in the larval rearing process and prevention of gut epithelium
scaling syndrome in the larvae. These products look promising for optimizing
P. schmitti postlarvae production.
USO DE BACTERIAS BENEFICIOSAS EN LA LARVICULTURA
DEL CAMARON Penaeus schmitti.
Resumen
Recientemente han aparecido en el mercado microorganismos
vivos que actúan como degradadores biológicos de resíduos
orgánicos en Acuicultura. Se probó el efecto de dos productos
en la cría de larvas del camarón blanco Penaeus schmitti,
que es cultivado a nivel comercial en Brasil y en Cuba. Se evaluaron dosis,
así como la acción de los productos sobre la calidad del agua
y sobre el posible control del síndrome de descamación del
epitelio del tracto digestivo de los animales, conocido como "Bolitas".
Se tomaron en cuenta la supervivencia, la velocidad de metamorfosis, la
calidad de las larvas y el crecimiento alcanzado hasta postlarva1. Se concluyó
que los productos pueden ser suministrados desde Protozoea I . Los mismos
posibilitaron la reducción de la tasa diaria de renovación
del agua de 100% para 30% en la larvicultura, además evitaron la
aparición del síndrome de descamación del epitelio
digestivo de las larvas. Estos productos presentan buenas perspectivas de
éxito actuando en la optimización de la producción
de postlarvas de P. schmitti.
As more intensive methods of larviculture have
been developed, the incidence of disease has increased. All referring publications
agree that the main method for avoiding disease requires the maintenance
of good handling of the larviculture together with strict sanitary control
and constant monitoring to maintain a balanced ecology
Until recently, the use of antibiotics was the
only method commonly practiced to control the undesirable bacteria. The
use of antibiotics creates its own difficulties, such as the associated
increase of bacterial resistance to antibiotics, increased ecological problems
caused by the presence of resistant bacteria, toxic chemistries of the antibiotics
and later, the restriction of which drugs are allowed to be used for shrimp
farming, necessitated the search for other biological options to control
the diseases. The alternative of being able to manipulate the bacterial
flora and the ecology of the systems of larviculture through the inoculation
of beneficial bacteria is very viable, so that the source of laboratory
postlarvae can continue to be established in the future.
The most novel option in this field is the use
of probiotics to manipulate the bacterial flora in the commercial laboratories.
According to Garriques and Arevalo (1995) several theories exist that explain
the performance of the probiotics: competitive exclusion of pathogenic bacteria
in the Aquaculture system; improvement of digestion by ingestion of essential
nutrients; improved nutrition by ingestion of essential enzymes; substances
released by bacteria, during their natural transformation of organic matter,
that inhibit the growth of opportunistic pathogens. This practice includes
the culture of selected stocks of beneficial bacteria that are inoculated
intentionally in the larviculture tanks.
Recently, products have appeared in the market
that combine bacteria able to degrade organic matter with nitrifying bacteria
that eliminate ammonium in the water. These products, are easy to manipulate
and store, in addition to possessing the desired properties, thus avoiding
the necessity of producing the bacterial cultures in the aquaculture laboratory,
reducing expenses of personnel and additional facilities.
Few references are available on the use of these
products in the culture of marine shrimps, but none are available for their
use with Penaeus schmitti.
ACKNOWLEDGEMENTS
We thank the Federal University of Santa Catarina
and the National Council of Development Cientíífico and Technolóógico
(NCPq) of Brazil, for the support of this investigation. The Planaqua Company
Aquicultura Technologia of Brazil and M. Silva de Salinas, Ecuador provided
the Alken Clear-Flo products for the tests.
MATERIALS AND METHODS
The commercial live microorganisms, Alken Clear-Flo
1000 and 1200 (from ALKEN-MURRAY CORPORATION, 1994) possess the following
characteristics: ACF 1000. - is a brown colored powder that contains three
species of Bacillus (B. subtilis, 2 strains: B. licheniformis
and B. polymyxa) able to produce a fast reduction of the excess of
food, lees, etc. ACF 1200. - is a liquid with a cloudy appearance containing
26 million bacterias/ml. It is a combination of Bacillus subtilis, Pseudomonas
aeruginosa, P. stutzeri, P. fluorescens and Escherichia hermanii,
that degrades the excess organic matter. In addition, ACF 1200 contains
the bacteria Nitrosomonas europaea and Nitrobacter winogradsky
for the elimination of ammonia and nitrite in water.
(Manufacturer's note: ACF 1200 was
improved in 1997, increasing the count to 500 million colony forming units
of bacteria/ml and P.aeruginosa was replaced by two strains of P.
putida, E. hermanii was replaced with Enterobacter cloacae and
P. stutzeri was replaced by B. licheniformis. The improvement
allows for a lower dose to be applied. ACF 1000 was improved in 1999 by
substituting B. thuringiensis for B.polymyxa)
Three experiments were conducted, first to evaluate
if the doses recommended for P. vannamei are adapted for larvae of
P. schmitti, without affecting the survival, the speed of metamorphosis
and the quality of the animals. The second test was made to determine the
action of the product on the quality of water, with rates of daily water
change reduced from 100% tor 30% for 100 densities of larvas/litro. A third
experiment was made in larviculture of HD to evaluate the effect of ACF
1000 before the presence of well-known diseases like " bolitas ",
primarily in the phase of protozoea.
Dosage of the products ACF 1000 and ACF 1200.
ACF products are recommended from the
beginning of the sub-stage of Protozoea II for P. vannamei in Ecuador.
The dose for 1000 ACF is 0.2 ppm initially, being decreased to 0.1 ppm every
day after the water is changed. If some problem is detected in the larviculture,
such as the presence of the disease "bolitas" or a high load of
organic matter in the tanks, an initial dose of 0.5 ppm is increased to
1 ppm in the following days (Silva R., Laboratory Brave Sea, Salt mines,
personal Ecuador, com., 1995). For ACF 1200, the manufacturer recommends
a daily dose of 5 ppm for high levels of organic matter; 2.75 ppm for average
levels and 0.5 ppm for low levels. This experiment proved that the doses
recommended for P. vannamei were correct in this case for use at
the end of Protozoea I. Fueron used cylinder-conical fiber glass tanks with
40 liter capacity. They count on a valve in the inferior part for the positioning
of the equipment of water interchange. The volume of water used was 30 liters.
The sea water used by the main network of the laboratory, previously filtered
by sand and cartridges of 5 micrometers, with influent temperature of 26
C, was obtained by means of heat diffusing equipment.
Through the experiment the water temperature
was controlled at that level by using 100 watt heaters in each experimental
unit, which are connected to a central thermostat. Fueron used Nauplios
III of desove, which they were selected by positive fototactismo and incubated
at the rate of 100 nauplios/liter. The applied experimental design appears
in Table 1. There were three processings with four simultaneous repetitions,
totalizing 12 experimental units.
TABLA 1. TRATAMIENTOS UTILIZADOS CON LOS PRODUCTOS ALKEN CLEAR-FLO
The rate of daily water exchange was 100% from
Protozoea II, with products added immediately after the water exchange of
each tank, with the exception of the first application in Protozoea I. The
feed consisted of diatoms (Chaetoceros calcitrans) from Protozoea
I that were adjusted three times a day to produce concentrations of 80 million
cells/ml and of flagellated (Tetraselmis tetrathele) that were offered
from Protozoea II. The biological parameters examined to analyze the results
were: daily survival, larval quality as determined by microscopic observations
twice a day and speed of growth, applying the parameters of Desarrollo de
Villegas and Kanazawa (1979).
Effects of the products Alken Clear-Flo on
the reduction of the rate of water exchange.
One function of these products is the control
of ammonium and the nitrite, in addition to the degradation of excess organic
matter present in the system, composed of leftover ration, dead lees, seaweed,
etc. This probably allows a considerable reduction of the rate of water
exchange, without harming the quality of the same one. In this test the
rate of renovation adopted throughout the period of experimentation was
of 30% as experimental conditions were equal to those of the previous experiment.
The experiment included the phases from Nauplio III to Postlarva1. From
Protozoea III the dose of products became stabilized in 0.5 ppm for ACF
1000 and 5 ppm for ACF 1200, added diaramente to the experimental units
after carried out the renovation of the water (30%). The registered physical
parameters were: temperature, pH and ammonium oxidized. The observed biological
parameters were: daily survival and speed of growth of the animals. In addition,
samples were viewed with the microscope to evaluate the larval quality during
the experiment. The feeding of the larvae followed the pattern used by the
Laboratory of Camarõões Marinhos-UFSC (Table 2).
TABLA 2. ALIMENTACION DE LAS LARVAS EN EL EXPERIMENTO DE LA
REDUCCION DE
LA TASA DE RENOVACION DE AGUA.
Evaluation of the effect of Alken Clear-Flo
1000 on the presence of "bolitas".
There was preliminary evidence of the effect
exerted on the well-known disease "bolitas" by the product ACF
1000, in the larviculture tanks of P.schmitti. In Ecuador, this product
is used as a prophylactic and curative in commercial systems raising the
larvae of P. vannamei.
Two fiber glass tanks were used with a volume
of 5 cubic meters, of white color and semicircular bottom. The tanks were
incubated to HD (400 nauplios/liter) with diverse originating spawning larvae
of the sector of maturation of the Laboratory. Filtered sea water was utilized
in the system of the laboratory, withe 34% solids and average temperature
of 26 °C. This test was developed from Nauplio III to Protozoea III,
since the mortality caused by this syndrome is commonly observed in the
sub-stage of Protozoea II.
The feeding consisted of the microalga
Chaetoceros calcitrans, maintained to a density of 80 thousand cel/ml
in the two experimental units, adjusting three times per day. From the end
of the sub-stage of Protozoea I, it was applied to the one of the tanks
processing with ACF 1000, in the initial concentration of 0.2 ppm, being
increased 0.1 ppm daily until the end of the experiment. To the second tank
no product was added, instead the methodology of culture of the Laboratory
was used. The larval survival was determined daily, and their state of health
was also observed microscopically three times per day, to verify quality
and the presence of "bolitas".
RESULTS AND DISCUSSION
Dosage of the products Alken Clear-Flo 1000
and1200.
The survival reached in Nauplio III to Protozoea
III was high, not difriendo of the pattern without products. In all of the
processings in which the survival was nearly 100%, differences in the consumption
of microalga by the larvae was observed, according to the verification of
the residual alga conducted three times per day. The quality of the larvae
between the processings was similar. The larvae stayed active, with positive
fototactismo and occupied digestive tract. The observation of fecal cords
showed that the food throughout was well digested during each one of the
processings of the experiment. The speed of growth of Nauplio III to Protozoea
III was similar for all the cases.
Based on these results it was possible to
state that the processings with Alken Clear-Flo 1000 and 1200 do not cause
mortality nor apparent damage to the larvae of P. schmitti during
the protozoea phase, when the peneidos are considered very delicate.
The doses used for P. vannamei in Ecuador from Protozoea II can be
applied in P. schmitti from Protozoea I.
The application of products from the end of this
sub-stage is considered to guarantee that the shrimp will not be affected
by such applications, providing therefore a greater security in the preventive
processing of the diseases, among them, the one of the syndrome of destruction
of the epithileal cells of the digestive tract that is pronounced in Protozoea
II.
Effect of the products Alken Clear-Flo on
the reduction of therate of water exchange.
The test values of total ammonium were below
the acceptable limit for shrimp larvae (0.10 to 0.75 g/liter), despite reducing
the rate at which water was changed from 100% to 30%, which appears to be
due to the positive effect of products on the quality of the water. In the
processing pattern the ammonium levels were superior, with little of the
white precipitate that indicates excess organics, according to the indications
of the kit used for the measurements. The test results indicated values
of pH between 7.5 and 8.0 for the three processings. At the end of the experiment,
there was superior survival of the larvae from Protozoea III treated with
Alken Clear-Flo when compared with that of the untreated larvae for postlarva1,
as can be observed in Table 3. These results demonstrate that a positive
effect of products existed during the larval phase.
TABLA 3. SUPERVIVENCIA ALCANZADA CON LOS PRODUCTOS ALKEN CLEAR-FLO.
In Table 4 it is possible to observe
that the growth of the animals was also different with the application of
products. A stature of 4.36 mm for postlarvae1 was obtained with ACF 1000,
that was significantly superior as compared to ACF 1200 from 4.22 mm and
to the pattern without products of 4.14 mm that were not different to each
other. This suggests product ACF 1000 also acts like food for the larvae.
TABLA 4. TALLAS MEDIAS DE LAS POSTLARVAS TRATADAS CON ALKEN
CLEAR-FLO.
It has been indicated that the growth of the
animals is generally determined by the feeding (Kuban and col., 1985; Alfonso
and col., 1988). Similar criteria indicate and col. Striking (1995) states
that the growth obtained for larvae of P. setiferus from the provided diet
was also affected by the physiological state of the animals. It is also
known that the bacteria comprise a portion of the shrimps feed, so that
this "pool" of bacteria is implicated as being responsible for
the increased growth of the shrimps obtained when ACF 1000 was added.
The quality of the larvae in the first
days (NIII - PIII) stayed similar according to the observations of their
conduct and the morphology. From Mysis I until Postlarva 1, the animals
of the experimental units pertaining to the processing pattern without products
showed dirt in the appendices, slow swimming and smaller response to the
light source. These signals are characteristic of the low rate of water
change, since in this phase there is necessity of major water changes above
30%. Nevertheless, the effect of products ACF 1000 and 1200, makes possible
smaller water changes as the bacteria degrade the organic matter present
in the water bodies. These products have a beneficial action on the quality
of the water, and consequently, on the survival, the quality of the larvae
and its growth.
Evaluation of the products Alken Clear-Flo
1000 against the presence of "bolitas".
The differences in the survival
of the Protozoeas dosed with ACF 1000 as compared with those that were not
treated:
Larvas treated: P II =100% P III = 97.5%:
Untreated larvas: P II = 94.3% P III = 56.05%
These results for P. schmitti are similar
to those found by Silva (in a personal communication) in commercial laboratories
of P. vannamei, using the same product. Through the microscope it
was observed that the treated larvae from Protozoea II did not display the
later portion of empty digestive tracts or dirt in the appendices. According
to the counts of the residual microalga in the tanks, the mean consumption
of the food also differed between treated and untreated:
The larvae without processing consumed less seaweed
in the presence of the disease, whereas the treated larvae always displayed
occupied digestive tracts and the lees showed the digested affluent food.
It was also observed that the treated larvae did not display a deficient
response to the compared light and low vitality like the untreated larvae,
which appeared with empty digestive tracts, a syndrome typically seen in
animals with the syndrome of descamation of the epithileum of the digestive
tract, according to reports Morales (1992) in P. vannamei.
These products exerted a positive effect
in the control of this disease caused by bacteria during the larval phase
of P. schmitti. The execution of tests for the postlarval stages
would be recommendable, as well as to experience with other products. For
the obtained results, these products prepared with combinations of beneficial
bacteria display good prospects for use in intensive processes of larviculture
of P. schmitti.
CONCLUSIONS
It is possible to apply live commercial organic
degrading microorganisms from Protozoea I ofPenaeus schmitti.
The products Alken Clear-Flo 1000 and 1200 make
it possible to reduce the rate of water exchange from 100% to 30% in the
young of the larvae to enhance population densities.
Use of these products avoided the appearance
of the disease of "bolitas: in protozoaes of P.schmitti in the tanks
of intensive larviculture.
REFERENCES
Alfonso, E., L. Martínez, R. Gelabert
y S. Leal (1988): Alimentación de larvas del camarón Penaeus
schmitti. I. Diatomeas y flagelados. Rev. Invest. Mar., 9 (1):47-58.
ALKEN-MURRAY CORPORATION (1994): Waste degrader
for Aquaculture. Product Information Bulletin, New York, pp: 1-8.
Gallardo, P. P., E. Alfonso, G. Gaxiola, L.
Soto y C. Rosas (1995): Feeding schedule for Penaeus setiferus larvae
based on diatoms (Chaetoceros ceratosporum), flagellates (Tetraselmis
chuii) and Artemia nauplii. Aquaculture 131: 239-252.
Garriques, D. y G. Arevalo (1995): An evaluation
of the production and use of a live bacterial isolate to manipulate the
microbial flora in the commercial production of Penaeus vannamei
postlarvae in Ecuador. In: Proceedings of the Special Session on Shrimp
Farming of the World Aquaculture Society (Aquaculture '95), Feb/95,
San Diego California, pp: 53-59.
Kuban, F. D., A. L. Lawrence y J. S. Wilkenfeld
(1985): Survival, metamorphosis and growth of larvae from four peneids species
fed six food combinations. Aquaculture, 47: 151-162.
Morales, I. (1992): Observaciones sobre el
síndrome de descamación del epitelio digestivo "Bolitas"
en larvas de Penaeus vannamei en Ecuador. Memorias del I Congreso
Ecuatoriano de Acuicultura, Guayaquil, Ecuador, pp: 203-208.
Villegas, C. y A. Kanazawa (1979): Relationship
between diet composition and growth of the zoeal and mysis stages of Penaeus
japonicus Bate. Fish Res. J. Philipp., 4:32-40.
