Estudios patobiologicos en poblaciones cultivadas del camaron de agua dulce Macrobrachium rosenbergii (De Man, 1879), Isla de Margarita, Venezuela.
Se realizaron estudios sobre ocurrencia de mortalidad y presencia de parasitos afectando larvas, postlarvas y adultos de Macrobrachium rosenbergii, cultivados en la Isla de Margarita, Venezuela. Se observo la presencia de ciliados peritricos coloniales identificados como Epistylis y Vorticella spp., asi como se confirmo la ocurrencia de los ciliados suctorios Acineta y Tokophrya spp. como ectocomensales o epibiontes. Estos organismos fueron encontrados en las branquias y exoesqueleto de los cuatro estadios de crecimiento examinados. Tambien se detecto la presencia tanto de algas cianofitas Anabaena y Lyngbya spp. como clorofitas Cosmarium y Ulothrix spp. Tricomas filamentosos pertenecientes a la bacteria Leucothrix sp. fueron encontrados en muestras de branquias y exoesqueleto de postlarvas y adultos. Los estudios bacteriologicos en larvas de los estadios II y III, asi como del agua utilizada para su levantamiento, revelaron la presencia de abundante flora bacteriana, con predominancia de representantes de los generos Aeromonas, Arthrobacter, Flavobacterium, Moraxella, Pseudomonas y Vibrio. Un estudio mas detallado de aislamientos de aeromonas, pseudomonas y vibrios resulto en las especies Aeromonas formicans, Pseudomonas alcaligenes y Vibrio an guillarum, respectivamente. El hongo imperfecto Fusarium sp. fue detectado en especimenes de postlarvas y adultos, siendo exitosamente aislado de las branquias en placas de agar dextrosa Sabouraud.
Palabras clave: Macrobrachium rosenbergii, patobiologia, infeccion, Venezuela.
Studies on the presence of diseases and parasites affecting larvae, post-larvae, juveniles and adults of Macrobrachium rosenbergii, cultured in Margarita Island, Venezuela, were undertaken. The presence of peritrichous colonial ciliates identified as Epistylis and Vorticella spp., and suctorians identified as Acineta and Tokophrya spp., occurring as ectocommensals or epibionts was confirmed. These organisms were encountered on the gills and exoskeleton of the four stages of growth examined. The presence of the cyanophyte algae Anabaena and Lyngbya spp., and the chlorophyte algae Cosmarium and Ulothrix spp., was also detected. Trichomes of filamentous bacteria identified as Leucothrix sp. were encountered in samples of the gills and exoskeleton of post-larvae and adults. Bacteriological studies on larvae from stages II and III, and of the water used for the larvae, revealed the presence of an abundant bacterial flora, in which representatives of the genera Aeromonas, Arthrobacter, Flavobacterium, Moraxella, Pseudomonas and Vibrio were present. A more detailed study of the aeromonads, pseudomonads and vibrios showed that the isolates belonged to the species Aeromonas formicans, Pseudomonas alcaligenes and Vibrio anguillarum, respectively. Fungi imperfecta identified as Fusarium sp., were detected in specimens of post-larvae and adults, and the organism was successfully isolated from the gills on Sabouraud dextrose agar plates.
Key words: Macrobrachium rosenbergii, pathobiology, infestation, Venezuela.
PATHOBIOLOGICAL STUDIES ON CULTURED POPULATIONS OF THE FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII (DE MAN, 1879), MARGARITA ISLAND, VENEZUELA
The palaeomonid genus Macrobrachium Bate, 1868 contains approximately 125 species, of which some are relatively small while others are capable of reaching more than 30 cm in length. These freshwater prawns are widely distributed in tropical and sub-tropical regions of the world, and several species of Macrobrachium are being cultured on a commercial basis [2, 31]. The aquaculture potential for the various species of Macrobrachium in Central and South America has been analyzed by New [29, 30]. This worker concluded that the greatest potential currently exists for M. rosenbergii (De Man, 1879), a South-East Asian species the production of which as an aquaculture activity is now generally available as a "technological package". Aguilera-Hernandez et ai.  have reported that M. rosenbergii is presently cultured in Argentina, Brasil, Colombia, Costa Rica, El Salvador, French Guiana, Guayana, Honduras, Mexico, Panama, Surinam and Venezuela, in Latin America.
In common with other types of aquaculture operations, diseases and parasites are of importance in the culture of Macrobrachium spp. [5, 22], The present research programme was undertaken with the aim of detecting the presence of ectocommensals or epibionts, internal parasites and fungi in populations of M. rosenbergii cultured in Margarita Island, Venezuela, and of studying the bacterial flora of the larval stages of development and of the water in which these were reared.
MATERIALS AND METHODS
Specimens of larvae, post-larvae, juveniles and adults of the species Macrobrachium rosenbergii were obtained from La Salle Foundation for Natural Sciences, Margarita Campus, Nueva Esparta State, Venezuela. Larvae and post-larvae were obtained from intensive hatchery culture with a 25% daily water exchange, while the juveniles and adults were cultured semi-intensively in concrete tanks with 8% daily water exchange. A total of 1,600 larvae, 1,600 post-larvae, 45 juveniles and 30 adults were examined to detect different types of epibionts, fungi, and parasites. Samples of Stage II and Stage III larvae, and samples of the water in which these had been reared, were taken to the Aquaculture Laboratory of the Instituto Oceanografico de Venezuela, of the Universidad de Oriente.
All of the animals were subjected to a careful clinical examination . For the detection of algae, and of bacterial and protozoan epibionts, larvae were gently macerated between two glass slides for microscopical examination (400X) as wet mount scrapings from the exoskeleton and appendages. Wet mounts from the gills, hepatopancreas and abdominal musculature were similarly examined in the case of juvenile and adult prawns. The relative degree of infestation with epibionts was determined as recommended by Bell et ai.  and Lightner ,
Bacteriological examinations were undertaken on larvae produced in 70 liters capacity aquaria containing brackish water (salinity =16%), and on samples of this water. A salinity of 16% was used in all of the media utilized. The larvae were retained on an 80 pm filter, and were repeatedly washed with sterile artificial aged sea water. Weighed 0.5 g aliquots were prepared and aseptically homogenized in sterile brackish water, from which decimal dilutions were prepared for inoculation in 0.1 ml quantities onto plates of nutrient agar, incubated at 26[grados]C for 48-96 hours. The water samples were used to prepare decimal dilutions and were treated in a similar manner. Bacterial colonies which subsequently developed were picked off and purified, and the individual isolates were identified on a basis of specific procedures [7, 12, 24, 25, 32, 36],
Attempts to isolate fungal pathogens detected were made using plates of Sabouraud dextrose agar (salinity = 1.6%), inoculated with pieces of gill tissue and with aliquots of the decimal dilutions prepared from the homogenized larvae.
The water quality was evaluated on a basis of temperature ([grados]C), pH, salinity (%), nitrates (ppm N-[N.sub.2]), nitrites (ppm N-N[O.sub.3]) and ammonium (ppm N-N[H.sub.3]), using samples obtained during the routine water monitoring of the experimental culture.
A high degree of infestation was determined on the gills and exoskeleton of the specimens examinated. These infestations included the Cyanophyte genera Anabaena and Lyngbya causing grades 2, 3 and 4 infestations in post-larvae (25%), juveniles (30%) and adults (60%), and the Chlorophyte genera Cosmarium and Ulothrix causing grades 2 and 3 infestations in post-larvae (30%), juveniles (50%) and adults (25%), TABLE I.
Trichomes of filamentous bacteria identified as Leucothrix sp. were found causing grades 2 and 4 infestations in the gills and exoskeleton of adults and post-larvae, TABLE II.
Colonial peritrichous ciliates of the genera Epistyiis and Vorticeiia, and suctorians of the genera Acineta and Tokophrya, were commonly detected as epibionts on the gills of post-larvae, juveniles and adults, with grades of infestation ranging from 3 to 4. The highest levels of infestation were detected in larvae (90%), and levels of infestation of 60% were found in post-larvae, juveniles and adults, TABLE II.
No endoparasitic protozoans or metazoans were encountered in any of the freshwater prawns examined during this study.
The results of the bacteriological studies on the larvae and the water are summarized in TABLE III. It is apparent that the bacterial flora was composed of the genera Acinetobacter, Alcaligenes, Arthrobacter, Aeromonas, Flavobacterium, Moraxella, Micrococcus, Photobacterium, Pseudomonas, Vibrio and enterobacteria. Isolates of Alcaligenes, Arthrobacter, Micrococcus and Photobacterium spp. were obtained from the water, but not from the larvae, whereas isolates of Aeromonas and Vibrio spp. were obtained from the larvae but not from the water.
The isolates of aeromonads, pseudomonads and vibrios were identified as the species Aeromonas formicans, Pseudomonas alcaligenes and Vibrio anguillarum ("V. parahaemolyticus") sensu Evelyn , which same comprised 43.33% of all isolates from the Macrobrachium rosenbergii larvae examined, TABLE IV.
Antibiotic sensitivity tests, using an impregnated disc method , showed that 50% of these strains of Aeromonas, Pseudomonas and Vibrio spp. were sensitive to penicillin, 85.71% to chloramphenicol, and 78.57% to neomycin, novobiocin and tetracycline, respectively.
Cases of mycotic invasion of the gills and appendages of adult prawns (50%), and of the exoskeleton of post-larvae (50%) were detected. In all of these cases, the affected animals showed the presence of grayish-white to yellowish-colored lesions. Canoe-shaped macroconidia were detected in wet preparations from the gills, and a tentative diagnosis of Fusarium sp. was made. The fungus was successfully isolated from the gills and surface lesions on plates of Sabouraud dextrose agar, on which it produced an abundant cotton-wool type of mycelium, with yellowish or purplish-colored areas in the mycelium; conidiospores with oblong or ovoid septate macroconidia were produced, and served to confirm the diagnosis as Fusarium sp.
The basic water quality parameters of the larval rearing tanks are given in TABLE V. It can be appreciated that there was an increase in certain of the chemical characteristics (nitrites, nitrates and ammonia), but no notable changes in the physicochemical characteristics, when compared to the optimum values recommended for Macrobrachium rosenbergii culture .
Diseases of aquatic organism, including decapod crustaceans, are not simple events but rather they occur as the result of subtle inter-actions between the animal, the aetiological agent and the environment. Wedemeyer  concluded that the environment is the least stable of these three factors, and unfavorable environmental changes can produce stress conditions which serve to make the animals more susceptible to infections or infestations by facultative pathogens. The occurrence of stress, therefore, is possibly the one single factor of greatest importance in relation to the occurrence of many types of infection and/or contagious diseases in prawns.
The findings of the present work indicate that the larval stages of Macrobrachium rosenbergii appear to be more susceptible to disease problems associated with mortalities than do the post-larvae, juveniles and adults. Similar observations were recorded by Goodwin & Hanson  in Hawaii (USA), who pointed out that the low survival rate of the larval stages frequently prevents exploitation of the full economic potential of the giant freshwater prawn. In the present programme, high larval mortalities occurred (~60%) possibly due, in part, to the presence of a heavy organic load in the water at the hatchery, which water appears to have contained numerous potentially pathogenic bacteria. Brock & Lightner  sustained that when the physico-chemical parameters of the water are within normal acceptable limits and no mortality due to parasites can be demonstrated, then facultatively pathogenic bacteria can often be shown to be the cause of such mortalities.
The bacteriological studies carried out on the larvae demonstrated the presence of three types of bacteria recognized as being potentially pathogenic to crustaceans and other aquatic animals, namely: Aeromonas formicans, Pseudomonas alcaligenes and Vibrio anguillarum (='V. parahaemolyticus"). The occurrence of such microorganisms in relation to freshwater prawn larvae represents a potentially dangerous situation in the survival of the Macrobrachium rosenbergii , V. anguillarum, in particular, is of special interest in view of the active chitinolytic properties detected in all of the isolates obtained during this work. Chitinoclastic or chitinolytic bacteria have been shown to be involved in a condition denominated "Shell Disease" in crustaceans [21, 22, 27], In the same way, A. formicans, Flavobacterium sp., Ps. piscicida and Vibrio spp., have been reported in association with aggregation and mortalities of penaeid shrimp zoeae and mysids in culture conditions [4, 6, 33]. The fact that the bacterial isolates should have shown a high degree of susceptibility to various antimicrobials indicates that these bacteria had not been in previous contact with antimicrobials and, indeed, such substances were never used in the rearing facilities.
A frequently occurring condition detected here was an extensive secondary infestation of the gills by algae, epibiont protozoans and filamentous bacteria, the presence of which gave rise to lethargy and lack of normal reflex movements in the affected prawns. Similar observations have been made by Smith et al.  in Macrobrachium rosenbergii cultured in the USA. As Brock [9, 10] has previously indicated, the occurrence of low numbers of epibionts has a minimal effect on the host, since these organisms do not penetrate the cuticle or cause a localized inflammatory response in the invaded area. Trichomes of Leucothrix sp. were also found causing moderate infestations in the gills and exoskeleton of post-larvae and adults. Although the presence of numerous filamentous bacteria was demonstrated, without any pathological changes being detected in the tissues, the degree of infestation could increase in periods of stress and lead to serious effects in the tissues due to secondary invasion by facultatively pathogenic eubacteria. Heavier infestations, however, are of greater importance, particularly in the case of the gills, where the infestation can impede normal respiration and give rise to mortalities associated with asphyxia or hypoxia [16, 38]. Infestations associated with ciliates of the epistylid genera Epistylis and Opercularia, the vorticellid genera Vorticella and Zoothamnium, and suctorians of the acinetid genera Acineta, Acinetides and Tokophrya, have been reported from adult Macrobrachium rosenbergii cultured in ponds in Limon Province, on the Caribbean coast of Costa Rica [14, 15, 23].
The detection of infections in Macrobrachium rosenbergii in Venezuela caused by Fusarium sp. is of interest. These cases were detected chiefly in adult prawns, which had previously undergone damage to the exoskeleton, although no mortalities from this cause were observed. Burns et al.  isolated this genus of Fungi imperfecti from M. rosenbergii and concluded that the fungus is not readily transmitted from diseased animals to healthy ones in the same facility. Infections due to Fusarium spp. have also been reported from penaeid shrimps [3, 34, 39].
On a basis of the results obtained during the execution of this study, the following recommendations have been made with reference to the prevention of disease problems in Macrobrachium rosenbergii culture operations in Venezuela or other tropical regions: (1) Careful attention must be given to maintaining optimum water quality parameters, with daily changes of at least 50% of the available water volume in hatchery facilities; (2) Uneaten food and other organic debris (including moulted shells) must be regularly removed from the water; (3) The population density of the prawns must be carefully monitored, and the numbers of animals maintained at an optimum level.
The authors wish to express their gratitude to the Aquaculture Department of the "La Salle Foundation for Natural Sciences", for the provision of specimens and facilities. They also thank to Faculty of Veterinary Sciences, Central University of Venezuela, for all the technical assistance made so freely available.
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Bethsy Rodriguez (1) Cesar Lodeiros (2), Gina Conroy (1,3), David Conroy (1,3) and Cesar Graziani (4)
(1) Seccion de Patobioiogia Acuatica, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, Apartado 4563. Maracay 2101-A, Edo. Aragua, Venezuela.
(2) Laboratorio de Acuicultura, Instituto Oceanografico de Venezuela, Universidad de Oriente. Cumana 6101, Venezuela.
(3) Unidad de Diagnostico y Asesoria Tecnica en Patologia Acuatica (UDAPTA)-Pharma-Fish SRL, Apartado 406. Maracay 2101-A, Edo. Aragua, Venezuela.
(4) Laboratorio de Camarones Dulceacuicolas, Departamento de Biologia, Universidad de Oriente. Cumana 6101, Venezuela.
Recibido: 17/10/2000. Aceptado: 09/03/2001.
TABLE I ALGAL EPIBIONTS DETECTED IN THE GILLS AND EXOSKELETON OF POST-LARVAE, JUVENILE AND ADULT Macrobrachium rosenbergii IN MARGARITA ISLAND, VENEZUELA Stage of Life Degree Algae Cycle of Infes- Localization % Animals Affected tation Affected CYANOPHYTA PL 2 Gills and 25 exoskeleton Order: Nostocales J 3 Gills and 30 exoskeleton Genera: Anabaena A 4 Gills and 60 and Lyngbya exoskeleton CHLOROPHYTA PL 2 Gills and 30 exoskeleton Orders: Zygnematales J 2 Gills and 50 and Ulotrichales exoskeleton Genera: Cosmarium A 3 Gills and 25 and Ulothrix exoskeleton PL = post-larvae. J = juvenile. A = adults. TABLE II PROTOZOAN AND FILAMENTOUS BACTERIAL EPIBIONTS DETECTED IN THE GILLS AND EXOSKELETON OF LARVAL, POST-LARVAE, JUVENILE AND ADULT Macrobrachium rosenbergii IN MARGARITA ISLAND, VENEZUELA Organism Stage of Life Cycle Degree of Affected Infestation Phyllum: CILIOPHORA L,PL,J,A 3 Class: Oligohymenophorea Sub-class: Peritrichia Order: Peritrichida Family: Vorticellidae Genus: Vorticella Family: Epistylidae L,PL,J,A 3 Genus: Epistylis Class: Kinetofragminophora L,PL,J,A 4 Sub-class: Suctoria Order: Suctorida Family: Acenetidae Genus: Acineta Genus: Tokophrya L,PL,J,A 3-4 Filamentous bacteria PL 4 A 2 Organism Localization on Host % Animals Affected Phyllum: CILIOPHORA Gills and exoskeleton 90 (L) Class: Oligohymenophorea (including appendages) 60 (PL) Sub-class: Peritrichia 60 (J,A) Order: Peritrichida Family: Vorticellidae Genus: Vorticella Family: Epistylidae Gills and exoskeleton 90 (L) Genus: Epistylis (including appendages) 60 (PL) 60 (J,A) Class: Kinetofragminophora Gills and exoskeleton 90 (L) Sub-class: Suctoria (including appendages) 60 (PL) Order: Suctorida 60 (J,A) Family: Acenetidae Genus: Acineta Genus: Tokophrya Gills and exoskeleton 90 (L) (including appendages) 60 (PL) 60 (J,A) Filamentous bacteria Gills and exoskeleton. 50 Gills 30 L = larvae. PL = post-larvae. J = juvenile. A = adults. TABLE III DISTRIBUTION (%) OF THE PRINCIPAL TYPES OF BACTERIA ISOLATED FROM Macrobrachium rosenbergii LARVAE AND FROM THE WATER IN WHICH THESE HAD BEEN REARED IN MARGARITA ISLAND, VENEZUELA Bacterial Type Larvae (%) Water (%) Acinetobacter 13.8 12.9 Aeromonas 20.6 - Alcaligenes - 3.2 Arthrobacter - 6.5 Enterobacteria 3.5 9.7 Flavobacterium 27.6 29.0 Micrococcus - 6.5 Moraxella 17.2 9.7 Photobacterium - 3.2 Pseudomonas 10.3 19.3 Vibrio 3.5 - Unidentified isolates 3.5 - TABLE IV PRINCIPAL CHARACTERISTICS OF POTENTIALLY PATHOGENIC BACTERIA ISOLATED FROM Macrobrachium rosenbergii IN MARGARITA ISLAND, VENEZUELA Caracter. Strain No. 1 2 3 4 5 6 7 Gram. - - - - - - - Morphology Rod Rod Rod Rod Rod Rod Rod Flagella P P P P P P P Ns flagella 2-3 1 1 1 1 1 1 Growth at 5[grados]C - - - - - - - 37[grados]C + + + + + + + 42[grados]C + + + + + + + Growth at 0% NaCl + + + + + + - 6% NaCl + + + + + + + 8% NaCl - + - - + + + 10% NaCl - + - - - - - Glucose O/F NR F F F F F F Gas from glucose - - - - - - - Acid from glucose - + + + + + + Acid from + + + + + + + saccharose Acid from lactose - + + + + + - Acid from mannose + - + + + + + Acid from - - - - - - + arabinose Arginine - - - - - - - dehydrolase Lysine - - - - - - + descarboxylase Ornithine - - - - - - + decarboxylase Gelatine - + + + + + + liquefaction Lipase production + + + + + + + Starch hydrolysis +/- + + + + + - Chitin hydrolysis ND + ND - - - + Oxidase + + + + + + + Indole production - + + + + + + Catalase + + + + + + + Methyl red - + + + + + + Nitrate reduction - + + + + + + Nitrate utilisation - + + + + + + Citrate utilisation + + - - + + + [H.sub.2]S production - + - - - - - O/129 sensitivity - - - +/- - - + Caracter. Strain No. 8 9 10 11 12 13 14 Gram. - - - - - - - Morphology Rod Rod Rod Rod Rod Rod Rod Flagella P NP P P P P P Ns flagella 1 NP 3 1-2 2 2-3 2 Growth at 5[grados]C - - - - - - - 37[grados]C + + + + + + + 42[grados]C + + + + + + + Growth at 0% NaCl + + + +/- + + + 6% NaCl + + + + + + + 8% NaCl - + + + + + + 10% NaCl - + + +/- + + + Glucose O/F F AL AL AL AL AL AL Gas from glucose - - - - - - - Acid from glucose + - - - - - - Acid from - - - - - - - saccharose Acid from lactose - - - - - - - Acid from mannose + - - - - - - Acid from + + +/- - - +/- +/- arabinose Arginine - + - - + - - dehydrolase Lysine - - - - - - - descarboxylase Ornithine +/- + +/- + + +/- +/- decarboxylase Gelatine + + + + + + + liquefaction Lipase production + + + + + + + Starch hydrolysis - - - - - - - Chitin hydrolysis - + - - - - +/- Oxidase + + + + + + + Indole production + - - - - - - Catalase + + + + + + + Methyl red - - - - - - - Nitrate reduction + - + + + + + Nitrate utilisation + - + + + + + Citrate utilisation + + - - + - + [H.sub.2]S production +/- - - - - - - O/129 sensitivity - - - - - - - +: positive. -: negative. +/-: weak positive. NR: not reactive. F: fermentation. AL: alkaline reaction. ND: not determined. TABLE V MEAN VALUES OF THE PHYSICO-CHEMICAL WATER QUALITY PARAMETERS IN THE CULTURE OF Macrobrachium rosenbergii IN MARGARITA ISLAND, VENEZUELA Stage of Life Temperature pH Salinity N-N[O.sub.2] Cycle ([degrees]C) (ppm) Larvae 25.2 8.3 12 0.6069 Post-larvae 25.2 8.5 0 0.5093 Juvenile 25.0 8.3 0 0.5133 Adults 25.7 8.0 0 0.6219 Stage of Life N-N[O.sub.3] N-N[H.sub.3] Cycle (ppm) (ppm) Larvae 0.7168 0.9532 Post-larvae 1.9001 1.9780 Juvenile 2.4059 2.0455 Adults 2.2334 2.3214
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|Author:||Rodriguez, Bethsy; Lodeiros, Cesar; Conroy, Gina; Conroy, David; Graziani, Cesar|
|Publication:||Revista Cientifica de la Facultad de Ciencias Veterinarias|
|Date:||Mar 1, 2001|
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