Aquaculture 174 Ž1999. 139–153 The use of microorganisms as food source for Penaeus paulensis larvae Fabiano L. Thompson, Paulo C. Abreu ) , Ronaldo Cavalli Department of Oceanography, UniÕersity of Rio Grande, Cx. P. 474, 96201-900, Rio Grande RS, Brazil Accepted 10 December 1998 Abstract Three experiments were conducted to test the usefulness of microorganisms as food source for the Penaeus paulensis larvae. Larvae fed only bacteria survived longer Ž3 days. than those cultured in filtered Ž- 1.0 mm. seawater. However, they grew better when fed flagellates and ciliates, reaching the protozoa II stage after 8 days. Gut content analysis showed that the ciliate Cristigera minuta was heavily grazed by the larvae. The addition of microalgae and Artemia sp. nauplii besides microorganisms did not result in increased survival. However, all larvae that received supplementary food in the form of flagellates and ciliates showed larger cephalotorax length than those in the control treatments. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Microorganisms; Shrimp; Larvae; Food 1. Introduction The culture of marine shrimp has shown an exceptional increase worldwide, mainly due to improvement of methods and techniques related to hatching and rearing of larvae ŽBarnabe, ´ 1990.. Among these, the use of antibiotics assured higher production of larvae due to the suppression of pathogenic bacteria ŽGarriques and Arevalo, 1995.. However, after some time, resistant microorganisms appear, demanding increasing amounts of antibiotics for its elimination ŽDixon, 1994.. In addition to the use of antibiotics, water exchange is also applied in order to reduce the abundance of pathogenic bacteria and maintain water quality. Nevertheless, this contributes to the lowering of microorganisms responsible for nutrient recycling in the ) Corresponding author. Tel.: q55-532-336509; fax: q55-532-336602; e-mail: docpca@super.furg.br 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 5 1 1 - 0 140 F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 system, deteriorating the water quality, and leading to the use of intensive water treatments and manipulations ŽMaeda, 1994.. More recently, studies have tested the use of microorganisms in the control of microbial infections in rearing larvae with promising results ŽMaeda, 1989; Moriarty, 1990; Nogami and Maeda, 1992; Maeda, 1994; Austin et al., 1995; Gil, 1995.. The addition of selected bacteria Žprobiotics. in tanks or ponds may control deleterious forms through: Ža. competitive exclusion of pathogenic bacteria; Žb. enhanced nutrition of larvae by supplying essential enzymes; and Žc. production of antibiotic substances that inhibit the growth of undesired cells ŽGarriques and Arevalo, 1995.. Similarly, the use of microbially matured water selects non-opportunistic bacteria and thus protects larvae from the proliferation of pathogenic bacteria ŽSkjermo et al., 1997.. Aside from the properties listed above, bacteria can also act as a food source for larvae either by direct consumption, or by fueling aquatic and benthic foodwebs in aquaculture systems. Such features have been well documented for natural ecosystems, following the seminal papers of Pomeroy Ž1974. and Azam et al. Ž1983. who showed the importance of microorganisms in the transfer of matter and energy through aquatic food chains. However, few studies have been conducted in order to evaluate the importance of microorganisms as food source for marine larvae ŽMoriarty et al., 1983; Maeda, 1989; Moriarty, 1990; Douillet and Langdon, 1994; Moriarty, 1997.. In this study, we tested the hypothesis of whether bacteria and protozoan Žflagellates and ciliates. enhance the survival of the critical first stages of the larvae of Penaeus paulensis ŽPerez-Farfante, 1967.. Our objective was to verify if the type and size of ´ microorganisms could influence the survival and growth of P. paulensis larvae. 2. Material and methods Three experiments conducted during this study used larvae Žnauplii VI. of the shrimp P. paulensis ŽPerez-Farfante, 1967.. The larvae were obtained in the laboratory using ´ adults from wild broodstock captured along the Southern Brazilian coast ŽSanta Catarina and Rio Grande do Sul, 288S–488W.. Maturation of the female was induced by unilateral ablation of the eyestalk, and manipulation of environmental conditions ŽMarchiori and Boff, 1983.. The larvae, used in each experiment came from the same spawn, and were kept in tanks with controlled temperature Ž278C. for 2 days until the nauplii VI stage was reached. During this period, there was no food supply, and the larvae subsisted on their own reserves. The experiments were begun with an initial density of 100 nauplii VIrl, in systems without water exchange, but with constant aeration. The seawater used in the experiments was previously filtered through a cartridge filter system ŽCuno filters y1.0 mm pore size.. 2.1. Experimental design (Fig. 1) 2.1.1. Experiment 1 Experiment 1 was conducted to verify if P. paulensis larvae could survive by feeding only on bacteria. The experiment was composed of two treatments, in duplicate Ž15 l F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 141 Fig. 1. Experimental design of the three experiments. each.. Treatment 1A Žcontrol. consisted of filtered seawater, while treatment 1B received an addition of nutrients Žglucose—C 6 H 12 O6 , ammonium—NH 4 Cl and phosphate—Na 2 HPO4 P 2H 2 O. making a C:N:P ratio of 1060:2:1. 2.1.2. Experiment 2 This experiment, comprising of five treatments Ž3 l in triplicate., was intended to test the survival of P. paulensis larvae feeding on larger microorganisms Žflagellates and ciliates.. Treatment 2A Žcontrol. consisted of filtered seawater. Treatments 2B and 2C received nutrients Žglucose, ammonium and phosphate. making C:N:P ratios of 530:80:5 and 1060:160:10, respectively. Treatments 2D and 2E were composed of enriched and aged seawater ŽEASW., i.e., 3 days before the beginning of the experiment glucose, ammonium and phosphate were added to a tank containing seawater ŽC:N:Ps 530:80:5.. It was kept under constant temperature Ž278C. and with aeration allowing the growth of bacteria but mainly flagellates and ciliates. Maximum densities of 3443 flagellatesrml and 810 ciliatesrml were reached during this period. Treatment 2D received half EASW and half filtered seawater by volume, whereas treatment 2E received only EASW. 2.1.3. Experiment 3 This experiment was designed to check the influence of bacteria and protozooplankton addition to standard larviculture practices, where microalgae and Artemia sp. nauplii 142 F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 are normally supplied as the main food sources. The treatments Ž3 l volume in triplicate. were designed as follows. Treatment 3A consisted of filtered seawater. Treatment 3B received half EASW and half filtered seawater Žby volume., whereas treatment 3C received only EASW. All treatments received the microalgae Chaetoceros calcitrans from the beginning to day 6, maintaining an abundance of 10 5 cellsrml in the tanks. Estimates of this microalgae abundance were made daily using Neubauer chambers and light microscope. After the fourth day newly hatched Artemia sp. nauplii Žcontinental strain, Prime Artemia, UT, USA. were offered at a density of 5 organismsrml. 2.1.4. Physical and chemical analysis Water temperature, salinity, and pH were monitored daily using mercury thermometer Ž"0.18C., optical refractometer Atago Ž"1. and pH meter ŽDigimed; "0.01.. Samples for dissolved inorganic nutrients Žammonia and phosphate. were collected at the beginning and end of each experiment. Ammonia was measured according to UNESCO Ž1983. and phosphate as described in Strickland and Parsons Ž1972.. 2.1.5. Enumeration of bacteria and protozooplankton For the determination of bacterial, flagellates and ciliates abundance, the treatments were sampled each 36 h in experiment 1, and daily during experiments 2 and 3. Aliquots of 20 ml were fixed with Lugol ŽThrondsen, 1978. and kept in stoppered glass flasks. For bacterial and flagellates counts, volumes of 0.5–1.0 ml were stained with the fluorochrome Acridine Orange and visualized under epifluorescence microscope ŽZeiss Axioplan. ŽHobbie et al., 1977.. The lugol present in the water was removed using one to two drops of 3.0% wrv sodium thiosulfate solution ŽNishino, 1986.. Ciliates abundance was determined by scanning the entire bottom of 10 ml Utermohl ¨ chambers using inverted microscope ŽUtermohl, ¨ 1958.. Ciliate species was identified according to Kahl Ž1933., Curds et al. Ž1983. and Carey Ž1992.. 2.1.6. LarÕae Larval survival was estimated by enumeration of organisms at the beginning and at the end of the experiments. The larval stage was determined, using light stereoscope microscope, according to Iwai Ž1978.. In the third experiment the length of cephalotorax of larvae was estimated in 90 organisms of each treatment. In all experiments the stomach contents of nine larvae per treatment, were observed using Acridine Orange and epifluorescence microscope. The larvae were fixed with a 4% vrv formalin solution. After this, their surface was cleaned by first submerging them in a 25% vrv ethanol solution for 10 min, followed by sonication Žultra-sound 20 kHz, 30 s, Cole Parmer Ultrasonic Homogenizer 4710.. They were later washed three times in filtered 4% vrv formalin solution and placed over a darkened Nuclepore polycarbonate filter Ž0.2 mm pore size. settled on a glass slide, to create a dark background. Each larvae was covered with 2–3 drops of a 0.1% wrv solution of Acridine Orange before dissection. The dissection was conducted under a stereoscope microscope using two dissection needles. Two cross sections were made close to the mouth and at the end of the abdomen. The stomach content extruded when a cover slip was pressed on the larvae. After 10 min, the extruded material was stained by the Acridine Orange and readily visible under epifluorescence microscope. Table 1 Temperature Ž8C., salinity, pH, ammonia and phosphate concentration ŽmM. values in each treatment during the three experiments Experiment 1 Experiment 2 Experiment 3 1A 1B 2A 2B 2C 2D 2E 3A 3B 3C 27.5 Ž"0.5. 30 Ž"0. 7.7 Ž"0.1. 27.5 Ž"0.5. 30 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. 27.0 Ž"1.0. 34 Ž"0. 7.7 Ž"0.1. Ammonia ( m M) Initial – 0.39 Final 1.91 9.04 a Ž"1.71. 9.28 a Ž"0.41. 109.04 b1 Ž"12.78. 39.52 ab2 Ž"3.36. 244.52 c1 Ž"33.56. 77.38 b2 Ž"17.50. 34.28 abd1 Ž"6.48. 24.76 a2 Ž"5.85. 24.76 ad Ž"5.85. 14.52 a Ž"1.86. 12.85a1 Ž"0.71. 409.28 2 Ž"30.07. 23.57 b1 Ž"2.29. 262.14 2 Ž"8.93. 32.14 c1 Ž"1.79. 323.09 2 Ž"51.32. 1.37 a1 Ž"0.02. 0.97 b2 Ž"0.01. 6.19 b Ž"0.27. 4.51a Ž"0.65. 10.5 c1 Ž"0.10. 7.52 c2 Ž"0.25. 1.94 a1 Ž"0.05. 4.79 a2 Ž"0.41. 2.73 d1 Ž"0.05. 4.51a2 Ž"0.02. 1.54 a1 Ž"0.01. 6.56 a2 Ž"0.05. 2.17 b1 Ž"0.01. 8.11b2 Ž"0.20. 2.79 c1 Ž"0.02. 7.38 ab2 Ž"0.54. Temp. Ž8C. Salinity pH – Phosphate ( m M) Initial – 0.17 Final 2.46 – F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 Treatments Different letters and numbers indicate statistical differences Ž P - 0.05. among treatments and time, respectively. Mean Ž"SE.. 143 144 F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 2.1.7. Statistical analysis Differences of microorganisms number among treatments in the first experiment were tested using t-test ŽSokal and Rohlf, 1969.. The results of larval survival, cephalotorax length and ammonia and phosphate concentration of experiments 2 and 3 were submitted to the ANOVA method ŽSokal and Rohlf, 1969.. When significant differences among treatments were found Ž P - 0.05., we applied the a posteriori Tukey test for differentiation of the groups ŽSokal and Rohlf, 1969.. 3. Results Table 1 summarizes the physical and chemical results of the three experiments. Temperature, salinity and pH were stable during the assays. Ammonia concentration increased in most treatments where there was no nutrient addition, or where ammonium and phosphate were added in low concentration in comparison to glucose Ž1B.. In experiment 2, lower ammonia concentration was measured at the end of the study period in enriched treatments, indicating a direct uptake by bacteria, while in experiment 3 there was an increase of this nutrient towards the end of the experiment, probably due to food addition and larvae excretion. Similarly, phosphate concentration was higher at the end of the third experiment in all treatments. In experiment 2, only treatments with EASW Ž2D and 2E. presented an increase of phosphate at the final phase, while the concentration of this element showed a reduction in the control Ž2A. and treatments with nutrient addition Ž2B and 2C.. Increase in phosphate concentration was observed at the end of the study period in treatment 1B of the first experiment. The results of experiment duration, developmental stage and percentage of surviving larvae are shown in Table 2. For experiment 3, the larvae cephalotorax length is also shown. The duration of experiments increased from the first to the last, as well as the percentage of larvae that were alive at the end of the assays. In the first experiment no larvae survived beyond the protozoea I stage, while in treatments 2D and 2E of second experiment, they reached the protozoea II stage. At the end of the third experiment all larvae in the three treatments were in the post-larvae I stage, and those larvae in treatments 3B and 3C showed larger cephalotorax length than that hatched in filtered seawater Ž3A. ŽTable 2. Ž P - 0.05.. In the first experiment the number of small free bacteria varied between 0.01 and 15.08 = 10 6rml with maximum values occurring in the enriched treatment 36 h after the beginning of the study ŽFig. 2.. Filamentous bacteria appeared in great numbers after three days in the containers that had nutrient addition Ž0.6–7.78 = 10 4rml.. Flagellates Ž0.49–8.17 = 10 4rml. and ciliates Ž0–6.62 = 10 3rml. showed a trend to increase towards the end of the experiment ŽFig. 2.. Highest increase in ciliate numbers occurred after 72 h when most larvae were dead. In this experiment, most of the larvae were impregnated with organic matter and microorganisms and, at the final period, they were observed dead in the bottom of the containers, or showing very low activity. Abundance of small free bacteria in the second experiment showed a significant increase in most treatments after 24 h Ž0.21–11.04 = 10 6rml; Fig. 3.. The flagellate abundance responded to the increase in bacteria, showing maximum values at day 2 Treatments Experiment 1 Experiment 2 1A 1B 2A 2B 2C 2D 2E 3A Experiment 3 3B 3C Experiment duration Ždays. Survival Ž%. 1 0 Development stage Cephalotorax size Žmm. PZ I – 3 15 Ž"0. PZ I – 4 0.33 Ž"0.99. PZ I – 4 2.97 Ž"0.58. PZ I – 4 1.1 Ž"0.58. PZ I – 8 3.96 Ž"3.96. PZ II – 8 2.64 Ž"0. PZ II – 10 90.5 Ž"5.85. PL I 1.458 a Ž"0.009. 10 79.2 Ž"6.56. PL I 1.569 b Ž"0.009. 10 85.7 Ž"7.82. PL I 1.569 b Ž"0.006. For experiment 3, the cephalotorax size is also shown Žmm.. Mean Ž"SE.. Different letters indicate statistical differences Ž P - 0.05. among treatments. F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 Table 2 Experiment duration Ždays., survival Ž%. and development stage reached by larvae during the three experiments 145 146 F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 Fig. 2. Abundance of small Žcoccus and rod shape. and filamentous bacteria, flagellates and ciliates ŽTreaments 1A and 1B. during the first experiment. Ž0–7.17 = 10 4rml., although the number of these organisms was kept low in the treatments with EASW ŽFig. 3—2D and 2E.. Ciliates abundance varied between 0 and F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 147 Fig. 3. Abundance of bacteria, flagellates and ciliates ŽTreatments 2A, 2B, 2C, 2D and 2E. during the second experiment. 11.48 = 10 3rml. The ciliate Cristigera minuta showed a rapid growth between days 1 and 2 in the treatments with nutrient addition Ž2B and 2C., and its number dropped sharply in the following 24 h. The larvae in all treatments were covered with organic matter and microorganisms, especially in the control Ž2A. and enriched treatments Ž2B and 2C.. At the end of the fourth day, most larvae of these treatments were concentrated 148 F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 at the bottom of the containers. However, the larvae of treatments 2D and 2E were swimming actively in the tanks, even though their bodies were covered by organic matter. In the third experiment, a significant increase in bacterial abundance was observed after the fourth day of experiment Ž0.5 = 10 6rml to 6.9 = 10 7rml. ŽFig. 4.. Since there was no significant differences in bacterial number among treatments ŽANOVA, P ) Fig. 4. Abundance of bacteria, flagellates and ciliates ŽTreatments 3A, 3B and 3C. during the third experiment. F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 149 0.05., and considering the fact that the rise in number occurred after adding Artemia to the tanks, we supposed that the water used to grow Artemia had a high number of bacteria. This was confirmed in later analysis of water samples from Artemia Ž3.99 = 10 7 bacteriarml.. Flagellates varied between 0.2 and 12.26 = 10 4 cellsrml, with maximum value observed in treatment 3A at day 6 ŽFig. 4.. Ciliates abundance Ž0–1.67 = 10 3rml. showed a decrease between days 0 and 1 and after the fourth day ŽFig. 4., probably related to the larvae consumption. Larvae of the three treatments were initially covered with organic matter and microorganisms. However, they were completely clean after undergoing molt. In all the experiments, the stomach of larvae were full of particles as well as bacteria, flagellates and ciliates. In the third experiment the larvae also showed C. calcitrans in their stomach. Some remains of Artemia were also observed. It was not possible to quantify the relative importance of the different food items to the nutrition of the shrimp larvae. 4. Discussion A major constraint in penaeid larviculture is the susceptibility to microbial infections. Live food, such as Artemia sp. nauplii, has been shown to be a source of potentially pathogenic bacteria ŽDehasque et al., 1991; Verdonck et al., 1994.. During the hatching of Artemia cysts, the abundance of bacteria can increase ca. 100-fold, when compared to the initial numbers. This bacterial population is hard to be removed even when nauplii are rinsed with seawater and freshwater ŽVerdonck et al., 1991.. However, hatching of pre-disinfected Artemia cysts presented low bacterial number ŽMerchie et al., 1997.. We observed that Artemia nauplii added to the tanks worked as a vector, aggregating more bacteria. However, we have no indication whether the added bacteria could cause any damage to the larvae, or actually function as an extra food source. The main pathogenic effect observed in this study was the presence of bacterial epibiosis, leading to the death of most larvae in experiment 1. Infestation of P. stylirostris larvae by epibionts bacteria, was mainly caused by the species Aeromonas formicans, Pseudomonas piscicida and FlaÕobacteria sp. ŽLewis et al., 1982.. The infestation was prevented by adding some antibiotics Žgentamycin, nalidixic acid and acridine. to the cultures. In our case, the supply of flagellates and ciliates Žexperiment 2. seems to have helped the larvae to survive and be active. Moreover, it was clear from the third experiment that the use of more energetic food Žmicroalgae and Artemia sp. nauplii. allowed the larvae to molt and get rid of the microbial fouling. Similar results were obtained in the production of P. Õanamei post-larvae ŽGarriques and Arevalo, 1995.. Our study clearly demonstrated that microorganisms can represent an important food source for P. paulensis larvae. It was shown that the larvae can survive longer by feeding only on bacteria, although much better survival and growth were obtained when larger microorganisms Žflagellates and ciliates. were included in the diet. Though bacteria represent an important food source, due to its higher N and P contents, their small size Ž0.5–1.5 mm length. may be a problem, since it is hardly 150 F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 retained by the feeding apparatus of the larvae. To be properly consumed, bacteria must form aggregates or be attached to particles ŽConover, 1982.. Moreover, not all bacterial macroconsumers are equipped with bacteriolytic enzymes that allow them to digest capsules and slime produced by these microorganisms ŽMoriarty, 1990.. Bacteria, on the other hand, are wildly consumed by nanoflagellates and small ciliated protozoan ŽWilliams, 1981; Sherr and Sherr, 1984., playing an important, though indirect, role in fueling benthic and planktonic food webs with energy and matter, which is transferred to higher trophic levels. Some past studies have shown the importance of bacteria for the growth of meiofauna, present in ponds, that were later consumed by penaeid shrimp ŽMoriarty et al., 1983; Allan et al., 1995.. Others have indicated that the growth of bacteria in tanks, after nutrient addition, gave rise to the increase of protozoan, which was consumed by crab Portunus tridentatus larvae ŽMaeda, 1988.. The growth of bacteria observed in this study Žbefore or during the experiments. clearly worked to enhance the availability of flagellates and ciliates which, in turn, improved the culture of P. paulensis larvae, with the production of bigger larvae. Flagellates and ciliates are an important food source for many organisms of the zooplankton, like shrimp larvae ŽPorter, 1984.. Their significance derives not only from their elevated abundance, but also from their biochemical composition, with higher amounts of nitrogen than carbon per cell. They also represent a significant reservoir of essential nutrients like polyunsaturated fatty acids, sterols and amino acids, that are essential for the growth of penaeid shrimp ŽStoecker and Capuzzo, 1990; Lim et al., 1997.. Moreover, the bigger size of protozoan, as well as their ability of locomotion, probably made them more interesting prey to the P. paulensis larvae. Nevertheless, the use of protozoan in larviculture is not utilized on a large scale. To our knowledge, only the study of Maeda Ž1989. reports the use of the ciliate Strombidium sulcatum as a food source for the larvae of P. monodon. The addition of this ciliate resulted in increasing survival and molt rates of the larvae. In this sense, we consider that the addition of the ciliate C. minuta found in our study, as a complementary food source, could improve the rearing conditions of P. paulensis larvae. This ciliate has a reasonable size Žca. 20–25 mm. and seems to be heavily consumed by the larvae, as indicated by the variability of its abundance during the experiments, and also by direct observation of the larval gut contents. Moreover, their rapid growth in quite simple conditions indicates that large scale cultivation of this ciliate could be easily obtained. The importance of flagellates and ciliates for the feeding of shrimp larvae lead us to discuss another point related to shrimp aquaculture: the water exchange. Water renewal has been largely used in order to diminish deleterious effects of physical, chemical and biological imbalance, caused by intensive rearing conditions like high organism density and food addition ŽBarnabe, ´ 1990.. Water exchange has been also suggested as an alternative to reduce the number of vibrionaceae bacteria during the cultivation of larvae and juvenile stages of P. paulensis ŽBarbosa and Capra, 1994.. However, recent studies have questioned this method and shown better rearing results when the water exchange rates were reduced ŽHopkins et al., 1995; Vinatea and Andreatta, 1997.. Higher survival, dry weight, and metamorphosis rates were found for P. paulensis larvae grown in static water renewal conditions. This result was associated with high F.L. Thompson et al.r Aquaculture 174 (1999) 139–153 151 bacterial abundance in the tanks ŽVinatea and Andreatta, 1997.. The authors considered that bacteria was probably participating in the P. paulensis feeding, or improving the water quality through nitrification process. No reference was made to the possible presence of protozoan. In the light of our results, we may speculate that the high shrimp survival and growth observed by Vinatea and Andreatta Ž1997. resulted not only from the increase in bacterial number, but also from the presence of protozoan in the tanks, if enough time was given for the growth of these microorganisms. It is important to note that the highest values of flagellates and ciliates in our study were observed 2 to 3 days after the beginning of the experiments, while bacteria reached maximum values in 24 h. Thus, it is likely that high water renewal will allow the growth of bacteria in tanks, but not of flagellates and ciliates, that have lower growth rates. If this is true, water renewal actually works against the larviculture success, since it increases the probability of pathogenic bacterial cells to occur, but does not allow the growth of protozoan, that could control the bacterial abundance by grazing and represent a complementary food source to the larvae. This hypothesis remains to be tested in future studies. The results of this study are preliminary, but they point out the importance of microorganisms, particularly flagellates and ciliates, in the culture of P. paulensis larvae. The Protozoa represents an important food complement, which may improve larvae production of commercially important species. Acknowledgements The authors are grateful to C. Odebrecht, B. Biddanda, W. Wasielesky and an anonymous referee for the comments on a previous version of this manuscript. We also thank the help of C. Odebrecht in the identification of the ciliate C. minuta. F. Thompson and P.C. Abreu were supported by the Conselho Nacional de DesenvolviŽCNPq.. This work was also supported with grants from mento Cientıfico e Tecnologico ´ ´ Fundaçao de Amparo a Pesquisa do Estado do Rio Grande do Sul ŽFAPERGS.. ˜ References Allan, G.F., Moriarty, D.J.W., Maguire, G.B., 1995. 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