Influence of biofilm on the production of

Aquacult Int https://doi.org/10.1007/s10499-018-0246-2

Influence of biofilm on the production of Farfantepenaeus paulensis in pens in the Patos Lagoon estuary Eduardo Luis Cupertino Ballester 1 & Fabrício Martins Dutra 1 & Tito Luís Pisseti 2 & Ronaldo Olivera Cavalli 2 & Paulo César Abreu 2 & Wilson Wasielesky Jr 2

Received: 8 August 2017 / Accepted: 28 January 2018 # Springer International Publishing AG, part of Springer Nature 2018

Abstract The objective of the present work was to determine the influence of artificial substrates that increase the area for biofilm development on the production performance of Farfantepenaeus paulensis juveniles in pens. Shrimp were stocked at a density of 20/m2 in pen structures (bottom area = 50 m2) that were installed in the Patos Lagoon estuary. Two treatments with three repetitions were analyzed, where artificial substrates (polyethylene nets—1-mm mesh size) were added to increase the area for biofilm development by 100%, and where no substrates were added. During the experimental period, the biomass and the composition of the biofilm were assessed. After 86 days of rearing, no significant differences were found in shrimp performance between the treatments (p > 0.05). However, the examination of the chlorophyll a, dry weight, and composition of the biofilm indicated that the shrimp were actively consuming the biofilm attached to the artificial substrates. Significant decreases in the abundances of nematodes > 500 μm after the 56th day and of tintinnids and rotifers between day 28 and day 42, indicated that the shrimp were selectively predating on these organisms. Moreover, a decrease in the chlorophyll a concentration in the biofilm suggests that the shrimp were consuming the microalgae. Although the increase in the area for biofilm development did not improve shrimp performance, the shrimp presented the highest growth rates when they consumed most of the biofilm microorganisms.

* Eduardo Luis Cupertino Ballester [email protected]

1

Laboratório de Carcinicultura, Programa de Pós-graduação em Aquicultura e Desenvolvimento Sustentável, Universidade Federal do Paraná, Setor Palotina, Bloco IV, Rua Pioneiro, 2153, Jardim Dallas, Palotina, Paraná 85950-000, Brazil

2

Instituto de Oceanografia, Programa de Pós-graduação em Aquicultura, Universidade Federal do Rio Grande, Rio Grande, Rio Grande do Sul, Brazil

Aquacult Int

Keywords Shrimp production . Pen production . Pink shrimp . Microorganisms . Artificial substrates

Introduction The pink shrimp Farfantepenaeus paulensis is a native species from South America that is distributed from Ilhéus, Brazil, 14° S, to the northeast coast of Argentina, Mar del Plata, 38° S (D’Incao 1991). This shrimp represents an important fishing resource in the southern and southeastern regions of Brazil and is a species with a greater value to artisanal fishing, which is carried out in the Patos Lagoon estuary (D’Incao and Reis 2002; Costa et al. 2008; Ruas et al. 2011). However, large oscillations in the volumes captured in the estuary have been registered (D’Incao and Reis 2002), which directly affects the community of artisanal fishermen living in the Patos Lagoon estuarine area (Wasielesky Jr et al. 2004). As an alternative to generation income for artisanal fishery communities, research has been carried out to determine the possibility of breeding shrimp in low-cost systems, such as pen enclosures assembled in the Patos Lagoon estuary (Wasielesky Jr et al. 2002). The results of the research were promising and indicated high productive potential in this type of system (Wasielesky Jr et al. 2011; Wasielesky Jr et al. 2004; Jensen et al. 2016; Krummenauer et al. 2016). In addition, it was demonstrated that the production in pens could be developed in this area without causing major environmental impacts (Soares et al. 2004; Poersch et al. 2007). As the proposed production system is directed at serving low-income communities, the reduction of production costs is extremely important to reach economic viability (Wasielesky Jr et al. 2004). It is known that in the production of shrimp, approximately 50% of the production costs are attributed to food expenses (Akiyama et al. 1992; Epp 2002). On the other hand, it has already been demonstrated that the natural food present in the production environment can supply a significant part of the nutritional requirements of shrimp (Anderson et al. 1987; Cuzon et al. 2004; Abreu et al. 2007; Dutra et al. 2016). The use of biofilm, which is defined as a community of microorganisms that form on submerged surfaces (Thompson et al. 2002), has been demonstrated to be an interesting way to provide higher quantity natural food for the shrimp produced (Bratvold and Browdy 2001; Moss and Moss 2004). A positive effect of the use of biofilm has already been demonstrated for the pink shrimp F. paulensis during the nursery phase (Thompson et al. 2002; Jensen et al. 2006; Ballester et al. 2007). However, studies have not demonstrated the advantages of the use of biofilm by this species of shrimp during the growout phase. Therefore, the objective of the present study was to determine the influence of the use of artificial substrates, which increase the area for biofilm development, in the performance of juveniles of the pink shrimp F. paulensis produced in pens installed in the Patos Lagoon estuary.

Material and methods The experiment was carried out in BSaco do Justino^ Bay (32° 03′ 55″ S, 052° 12′ 30″ W), Patos Lagoon estuary, Rio Grande, RS, Brazil, over 86 days. Six round pens with 50 m2 bottom areas were assembled in the bay. The pen enclosures were made of PVC-coated polyester nets with a mesh size of 5 mm. The pen heights were 2.1 m (average depth of 0.80 m), and the frame structures were constructed of bamboo poles.

Aquacult Int

The experimental design was completely randomized and was composed of two treatments and three replicates. The treatment with substrates (S) included the addition of 25 m2 of artificial substrate (polyethylene net, 1.0-mm mesh size), which doubled of biofilm fixation. The artificial substrates were placed vertically in the central areas of the pens. The substrates were placed in the water 25 days before the shrimp were stocked to allow for the development of biofilm (Preto et al. 2005). The treatment without substrates (NS) were the pens without artificial substrate. Shrimp juveniles were supplied by the Marine Aquaculture Station of FURG—Federal University of Rio Grande, according to the methodology for the production of post-larvae described by Fast and Lester (1992). When the shrimp reached 29 days after metamorphosis to post-larvae (PL29), they were acclimated to the temperature (25.4 °C) and salinity (12‰) conditions in the estuarine bay and transferred to the nursery cages where they were stocked at a density of 320 post-larvae/m2. The animals remained in these structures until reaching weights over 0.36 g, which allowed for their stocking in the growout structures without the risk of escape through the 5 mm mesh (Jensen et al. 2004). During the 43 day nursery period, the prawns were fed four times daily with commercial feed (Purina®, 40% crude protein). When relocated to the growout pens, the average shrimp weight was 0.80 ± 0.28 g, and the animals were stocked at a density of 20 juveniles/m2. During the experimental period, the shrimp were fed twice each day. As the feeding activity of F. paulensis is higher at night (Quaresma and Sugai 2005), 1/3 of the daily amount of food was provided at 8:00 am, and 2/3 was provided at 9:00 pm. Initially, the feeding rate was 10% of the total shrimp biomass (Wasielesky Jr et al. 2011) and was adjusted throughout the experiment according to the food consumption observed in feeding trays (one tray 40 cm in diameter per experimental unit). During the first week of the experiment, the shrimp were fed the same diet used in the nursery phase. In the following week, the diet consisted of a mixture of the nursery and growout feed (Purina®, 35% crude protein). From the third week onwards, the feeding consisted exclusively of the growout diet. At the end of the experiment, the feed conversion rate (FCR) was calculated based on the survival, biomass gain and total feed offered in each experimental unit. Every 14 days, 30 shrimp from each experimental unit were randomly sampled, and their wet weights were registered to the nearest 0.01 g using a digital scale. After weighing, the shrimp were returned to their respective pens. At the end of the experiment, all shrimp were collected, counted and weighed to determine the survival, specific growth rate and final biomass of each experimental unit. The water temperature (8:00 a.m. and 5:00 p.m.—mercury thermometer, accuracy of 0.5 °C), salinity (Atago® manual refractometer, accuracy of 1‰) and transparency (Secchi disk, accuracy of 5 cm) were registered daily at a point located 5 m from the pens. The dissolved oxygen (Handylab OX1/Set SCHOTT® oximeter) and pH (Handylab 2 BNC SCHOTT® pH meter) were registered once each week (8:00 a.m. and 5:00 p.m.) at a control point 200 m from the production area and at a central point inside the pens. At these same sites, water samples were collected every 5 days to determine the total ammonia concentration (UNESCO 1983). Every 14 days, nine 2 × 2 cm fragments of the artificial substrates from treatment S were removed to determine the composition of microorganisms (n = 3), chlorophyll a concentration (n = 3) and biofilm dry weight (n = 3). The chlorophyll a concentration in the biofilm was determined through spectrophotometry (Strickland and Parsons 1972) using the equations proposed by Jeffrey and Humphrey (1975).

Aquacult Int

The extraction of the photosynthetic pigment was performed in 20 mL vials containing 90% acetone (Merck® PA) maintained in the dark at − 12 °C for 24 h. To evaluate the biofilm, dry weight samples were oven dried at 60 °C until a constant weight (approximately 24 h). The weighing was performed using a digital analytical scale with an accuracy of 0.0001 g (Sartorius®, model MC1 Analytic 210S). To characterize and count the microorganisms present in the biofilm, samples of the substrates were fixed in 4% buffered formalin solution. To remove the biofilm from the substrates, an ultrasonic device (Ultrasonic Homogenizer 4710 Series, ColeParmer Instrument Co.) was used at an amplitude of 20 Khz, 6 to 8 times in 15- to 20-s intervals followed by 15 to 20 s of rest to avoid heating the sample (Thompson et al. 2002). To count the bacteria and flagellates, 0.1-mL subsamples were filtered on polycarbonate membranes (Nuclepore—0.2-μm pore size, 25 mm diameter) previously darkened with irgalan black and stained with the DAPI fluorochrome at a final concentration of 10 μg/mL (Porter and Feig 1980). The counts were performed on a Zeiss Axioplan epifluorescence microscope, equipped with light filter 487,701 (BP365/11; FT 395; LP 397), with a final magnification of × 1000. The counts were performed in 30 fields per randomly selected blade. To count the diatoms, cyanobacteria, nematodes, copepods, copepodites, ciliates (tintinnids and vorticellids), and rotifers present in the biofilm, subsamples of 0.1 to 2.1 mL were placed in a sedimentation camera. The counting was performed on an inverted microscope Zeiss Axiovert equipped with phase contrast; the final magnification used was × 200 to × 400. A minimum of 30 fields per camera were randomly selected and counted (Utermöhl 1958). The identification of the most abundant genera of diatoms and cyanobacteria present in the biofilm was performed, when possible, according to Round et al. (1990) and Komárek and Anagnostidis (2005). The data on survival, final weight, final biomass and feed conversion rate of the shrimp produced were submitted to a Bt^ test (Sokal and Rohlf 1969) to verify the differences between the treatments. The specific growth rates of the shrimp, the chlorophyll a concentrations, the dry weights and the abundances of microorganisms in the biofilm were submitted to analysis of variance (ANOVA); when significant variations were found the Tukey test (α = 0.05) was applied to compare the averages. When the analyzed data did not obey the necessary assumptions for the use of the Bt^ test or the ANOVA, mathematical transformations were performed (log (x) or log (x + 1)). The relationship between the concentration of chlorophyll a and the dry weight of the biofilm was determined using the Pearson correlation analysis (Snedecor and Cochran 1982).

Results The mean temperature of the water in the production area declined throughout the experiment, with mean values of approximately 26 °C in the first 40 experimental days. In the second half of the experimental period, the averages declined to approximately 24 °C. The salinity presented an inverse effect, with mean values of 12‰ in the first half of the study and 18‰ in the second half. The water transparency values were stable throughout the experiment with an average transparency of 60 cm. (Table 1). The dissolved oxygen values were below what is recommended for shrimp production (2.2 mg/L−1) (Van Wyk and Scarpa 1999) only on the last day of the experiment. The pH values remained stable and were approximately of 8 throughout the experimental period.

Aquacult Int Table 1 Mean (± SD), minimum and maximum values of temperature (°C), salinity (‰) and water transparency (cm) during the production period of pink shrimp F. paulensis in pens in the Patos Lagoon estuary Variable

Mean (±dp)

Minimum

Maximum

Temperature at 8:00 Temperature at 17:00 Salinity Transparency

23.9 ± 1.24 26.9 ± 2.03 15.26 ± 7.57 63.43 ± 13.46

21 21 3 40

27 32 23 90

Variations between the control point and the production point were not observed on the days of measurement. The same occurred for the ammonia concentrations, with maximum values of 0.08 mg/L−1, thus below the levels considered harmful for this species (Ostrensky and Wasielesky 1995). Therefore, no significant differences (p > 0.05) were determined between control and production points for these variables (Table 2). The biofilm that formed on the artificial substrates was mainly composed of diatoms, filamentous cyanobacteria, bacteria, flagellates, ciliates, copepods, copepodites, rotifers, and nematodes. Among the diatoms found in the biofilm, the most abundant were of the Melosira, Surirela, and Pleurosigma genera. The most representative genera for cyanobacteria were Leptolyngbia, Phormidium, and Homolothrix. The ciliates present were vorticellids and tintinnids, whereas the copepods were from the Harpacticoides order. Nematodes of the genera Axonolaimus, Talassomonhystera, and Theristus were identified. The nematodes belonging to these genera are classified as non-selective depository eaters. The variations in the concentrations of chlorophyll a and the dry weights of the biofilm are presented in Fig. 1. The Pearson correlation between these variables had a coefficient of r = 0.71. The cyanobacteria and diatoms found in the biofilm were the main organisms that contributed to the autotrophic biomass. The chlorophyll a and dry weight analysis revealed a similar pattern for the two variables. Both variables presented a constant increase over the experimental period, and significantly higher (p < 0.05) values were observed on day 42 of the experiment. This significant difference was maintained until the 56th day, and on the 70th day of the experiment, a significant drop in these values (p < 0.05) was recorded. After, the variables returned to significantly increased (p < 0.05) until the end of the experimental period (86th day). The variations in the densities of bacteria and flagellates present in the biofilm are shown in Figs. 2 and 3, respectively. These microorganisms have similar patterns of variation. The bacteria presented significantly higher values (p < 0.05) on the 28th day of the experiment, and Table 2 Mean (± SD), minimum and maximum values of dissolved oxygen (mg/L−1), pH and total ammonia (mg/L−1) during the production period of pink shrimp F. paulensis in pens in the Patos Lagoon estuary Variable

Hour

Mean (±DP)

Minimum

Dissolved oxygen (pen) Dissolved oxygen (control) Dissolved oxygen (pen) Dissolved oxygen (control) pH

08:00

6.0 ± 2.3 6.8 ± 1.6 10.7 ± 2.1 10.9 ± 2.2 8.2 ± 0.4 8.7 ± 0.2 0.01 ± 0.03

2.2 4.5 8.0 8.0 7.6 8.4 nd

Total ammonia

17:00 08:00 17:00 –

nd not detectable by the analysis method

Maximum 9.9 9.9 11.7 11.7 8.7 9.2 0.08

Aquacult Int

Chlorophyll a and the dry weight of the biofilm

22

a

Dry weight (mg/cm2) Chlorophyll a (ug/cm2)

20

a

ab

18

bc c

16 c

14 12

b

b

10 d

8 c

6 4

c

d

c

c

2 0 0

14

28

42

56

70

86

Days Fig. 1 Mean values (± SE) of the variation in chlorophyll a and the dry weight of the biofilm formed on artificial substrates installed in the pens used for the production of F. paulensis (treatment S)

the highest density of the experimental period was recorded on the 42nd day (29.24 × 106/ cm2). Significant reductions in the abundance of bacteria were recorded on the 56th and 70th 32000000 a

30000000 28000000 26000000

b

24000000 Bacteria/cm2

22000000 20000000 18000000 16000000 14000000

c

c

12000000 10000000 8000000

d

d

0

14

d

6000000 4000000 28

42

56

70

86

Days Fig. 2 Mean values (± SE) of the variation in the density of bacteria adhered to the biofilm on artificial substrates installed in the pens used for the production of F. paulensis (treatment S)

Aquacult Int

120000 a

110000 100000

Flagellates/cm2

90000 80000 70000 b

60000 50000 40000

c

bc

bc c c

30000 20000 0

14

28

42

56

70

86

Days Fig. 3 Mean values (± SE) of the variation in the density of flagellates present in the biofilm on artificial substrates installed in the pens used for the production of F. paulensis (treatment S)

days. A significant increase was recorded on the last day of the experimental period. The flagellates presented a significant increase in their density on the 42nd day, followed by reductions in density on the 56th and 70th days. After this period, the density of the flagellates increased again on the last day of the experimental period, when it reached the maximum value (10.4 × 104/cm2). The variations in the densities of copepods, copepodites, tintinnids, vorticellids, rotifers, and ciliary cysts that were found in the biofilm are shown in Fig. 4. A significant reduction in the number of tintinnids, vorticellids, ciliates and rotifers were recorded between days 28 and 42. Between days 56 and 70, the number of tintinnids and rotifers again significantly declined. Although variations in copepod and copepodite densities were recorded, no significant differences were detected in their densities throughout the experiment (p > 0.05). A significant reduction in the number of nematodes over 500-μm length was observed after the 42nd day (Fig. 5). The same was not observed for the nematodes < 500 μm, which presented a considerable increase in abundance after the 28th experimental day (Fig. 5). After the 56th day of the experiment, significant reductions were observed in the number of nematodes > 500 μm in length and the number of tintinnids and rotifers (Figs. 4 and 5). The reduction of the density of these microorganisms was accompanied by the reduction in the dry weight of the biofilm in the same period (Fig. 1). After this period, significant increases (p < 0.05) in the densities of bacteria, flagellates, nematodes < 500 μm and rotifers were recorded, which coincides with the increase in the dry weight of the biofilm that was recorded between days 70 and 86. At the end of the experiment, no differences were observed (p > 0.05) between the final weight, survival, feed conversion rate and biomass values for the shrimp produced in the treatments with and without artificial substrate (Table 3). Figure 6 shows the specific growth

Aquacult Int 700 600

ab

400

bc

bcd

b

db

c

ab

ab

c b

100

ab

ab

a

abc

200

bc

a

a

b

b

b

42

56

cd

300

bc

Density/cm2

500

a

Copepods Copepodits Tintinnids Vorticelideos Rotifers Cysts of ciliates

b

db

b

0 0

14

28

70

86

Days

Fig. 4 Mean values (± SE) of the variations in the densities of copepods, copepodites, tintinnids, vorticellids, rotifers and ciliated cysts found in the biofilm formed on artificial substrates installed in the pens used for the production of F. paulensis (treatment S). The letters represent difference (p < 0.05) of each organism during the time

rates of the shrimp throughout the study period. Significant differences were not found between the specific growth rates of the shrimp produced in the two treatments (p > 0.05). However, both treatments presented growth rates higher between days 42 to 70 (Fig. 6). 1000

a

Nematodeos (total) Nematodeos>500um

900

ab

800

Nematodeos/cm 2

700

ab

bc 600 500

a 400

cd

cd

300

b

d bc

200

bc

0

bc

bc

100 0

14

28

42

c 56

70

86

Days

Fig. 5 Mean values (± SE) of the variation in the densities of the total number of nematodes and the number of nematodes with length > 500 μm found in the biofilm formed on artificial substrates installed in the pens used for the production of F paulensis (treatment S)

Aquacult Int Table 3 Mean values (± SD) of final weight (g), survival (%), final biomass (g/m2), feed conversion rate (FCR), and specific growth rate (SGR) from the production of F. paulensis in pens with and without artificial substrate over 86 days Treatment

Final weight

Survival

Biomass

FCR

SGR

With substrate (S) Without substrate (NS)

11.17 ± 1.72 10.99 ± 1.87

79.4 ± 3.0 85.6 ± 10.1

177.3 ± 6.2 187.3 ± 14.4

1.63 ± 0.18 1.65 ± 0.18

12.06 ± 2.00 11.85 ± 2.17

Discussion The water quality was considered acceptable for the production of the pink shrimp F. paulensis. Although a dissolved oxygen concentration below the recommended range for the production of penaeid shrimps was recorded (Van Wyk and Scarpa 1999), these records were brief and the performance of the shrimp was considered satisfactory when compared to other studies performed under similar production conditions (Wasielesky Jr et al. 2001; Soares et al. 2004). The results of the growth and survival of the shrimp in this study confirm the viability of the production of F. paulensis in pens in the Patos Lagoon estuary. In 86 days, the shrimp reached commercial size (approximately 11 g), with survival and feed conversion rates comparable to other studies performed with penaeid shrimp (Peixoto et al. 2003; Krumenauer et al. 2010). The use of artificial substrates to increase the biofilm growth area did not result in an increase in the shrimp performance between the treatments. This finding could be explained by the low production density employed in this study. Moreover, the substrates of the pens 3.0 S NS 2.5

(g)

2.0

1.5

Specific growth rate

1.0

0.5

14

28

42

56

70

86

Days Fig. 6 Mean values (± SE) of the specific growth rate of F. paulensis juveniles produced in pens with (S) and without (NS) artificial substrates over 86 days

Aquacult Int

themselves (polyester mesh) could have provided an area for the development of biofilm in the treatment without additional artificial substrates, thus providing biofilm for the consumption of the shrimp. Previous studies that have focused on the use of artificial substrates to increase the amount of biofilm and improve the performance of produced shrimp have had variable results. Bratvold and Browdy (2001) and Otoshi et al. (2006) obtained higher productivity during the production of juvenile of Litopenaeus vannamei when using artificial substrates (Aquamats®), while Samocha et al. (1993) observed no increase in the survival or growth of the L. vannamei post-larvae produced in the presence of extra substrates. Kumlu et al. (2001) reported significantly lower growth (p < 0.05) in Metapenaeus monoceros produced in aquariums, with a 200% increase in the area of biofilm growth in relation to the control treatment. According to Bratvold and Browdy (2001), the presence of substrates provides food for the shrimp in the form of biofilm and allows for a better distribution of the shrimp in the production tanks, thereby reducing the negative effects on survival and shrimp growth of increasing the stocking density. In the present study, the stocking density was relatively low (20 shrimps/m2). Therefore, it was not possible to verify the effect of the presence of substrates on shrimp distribution provided by the use of the substrates. However, in a previous study that carried out with post-larvae of F. paulensis produced at a density of 300 shrimp/m2 in pens installed in the same area of the current study found that the presence of biofilm and artificial substrates significantly increased the performance of shrimp (Ballester et al. 2007). Previous studies on the diet of F. paulensis have demonstrated that these shrimp consume microalgae, macroalgae, mollusks, polychaetes, small crustaceans in addition to organic matter, debris, and sediment (Soares et al. 2004; Soares et al. 2005). Soares et al. (2005) demonstrated that benthic organisms such as polychaetes and tanaidacea are among the items preferentially ingested by F. paulensis produced in pens. However, it has been shown that during shrimp production, the density of benthic invertebrates rapidly reduces over periods ranging from three to 8 weeks (Allan et al. 1995; Soares et al. 2005). In the present study, the decline in the abundance of biofilm microorganisms was more evident around the sixth week of production. A reduction in the total biomass of the biofilm formed on the substrates was also recorded around this time and was indicated by the chlorophyll a values and the dry weight of the biofilm. These changes in biomass and biofilm composition are an indication that the shrimp began searching there for the food necessary for development after they had already consumed the benthic organisms available. The subsequent increase in the biofilm biomass recorded between days 70 and 86 is probably due to the recovery of the microorganism communities that occurred due to the recruitment from the adjacent natural environment and the presence of vacant niches due to the reduction in the abundance of organisms present in the biofilm. Although the performance of the shrimp was not affected by the higher amount of biofilm available in treatment S, there was a considerable increase in the specific growth rates of the shrimp produced in the two treatments during two periods (42 to 70). These increases coincided with a significant reduction in certain groups of microorganisms in the biofilm (Fig. 6). Variations in the microorganism densities recorded during the experiment may have been caused by environmental variations or the trophic relationships between the different groups of microorganisms. However, it is more likely that the selective

Aquacult Int

predation of the shrimp was the main factor that determined the reduction in the abundance of certain microorganisms in the biofilm. The variation pattern in the bacterial and flagellate density followed the growth of the dry weight of the biofilm until the 42nd day of the experiment. However, from this date, the dry weight of the biofilm continued to rise, while the densities of the bacteria and the flagellates decreased, which indicated that other organisms might have consumed bacteria and flagellates. The variations between flagellates and bacteria densities were closely related, but this is probably the result of trophic interactions. It should be noted that bacteria can be predated upon by other microorganisms in the biofilm, such as ciliates and nematodes. In addition, flagellates also suffer from predation by ciliates and rotifers. Nematodes < 500 μm had the greatest growth between days 42 and 56, and these organisms are deposit eaters and possible consumers of bacteria. Between days 56 and 70, significant reductions in the numbers of rotifers, tintinnids and nematodes (> 500 μm) were observed, which indicates a possible selectivity of these organisms by the shrimp. Likewise, between days 28 and 42, significant reductions in the densities of rotifers, vorticellids, tintinnids and cysts of ciliates present in the biofilm were observed. From the 42nd experimental day, significant reductions in the number of flagellates and bacteria were observed. These microorganisms had been previously identified parts of the shrimp diet in natural environments (Allan and Maguire 1992; Allan et al. 1995; Nunes et al. 1997). In addition, the reduction in the chlorophyll a concentration of the biofilm also pointed to a consumption of the microalgae present in the biofilm. In previous studies, the selectivity of the post-larvae of F. paulensis was observed due to the consumption of diatoms in the biofilm (Preto et al. 2005; Ballester et al. 2007). Previous studies have demonstrated the nutritional contribution of microorganisms present in biofilm. Silva et al. (2008) determined that bacteria, filamentous cyanobacteria, flagellates and nematodes in biofilm are an important source of lipids, and diatoms and nematodes significantly contribute to the protein content of the biofilm. Schlechtriem et al. (2004) demonstrated that nematodes may be an important food source for carp larvae and emphasized their importance as a source of polyunsaturated fatty acids. Zhukova and Kharlamenko (1999) reported that flagellates and ciliates are able to synthesize long-chain polyunsaturated fatty acids from unsaturated fatty acids found in bacteria. The results of these studies demonstrate that the consumption of these microorganisms by the shrimp can add highquality nutrients of easy digestibility to their diet. Therefore, the presence of substrates for biofilm formation in the production environment would be a way to stimulate the growth of these microorganisms and make them available for shrimp consumption.

Conclusions The results of this study indicated that shrimp used biofilm as a food source, and the abundance of certain microorganisms of high nutritional value that were present in the biofilm community was significantly reduced throughout the experiment. Coincidentally, this reduction was notable during the period in which, as determined by previous studies, a reduction of the benthic organisms occurs, which are important resources consumed by shrimp. However, the 100% increase in the area available for biofilm development did not result in an improved performance of the shrimp produced. The presence of the substrates in the production pens (where biofilm growth naturally occurs) and the low stocking density of the shrimp used in this

Aquacult Int

study may have reduced the benefit of the use of extra substrates to provide a greater amount of biofilm. Therefore, it is recommended that studies using larger stocking densities should be performed, as the use of artificial substrates and biofilm may improve the performance of F. paulensis. Acknowledgements The authors would like to thank the researchers Clarisse Odebrecht, Marli Bergesh, and Taciana Cramer for their assistance in the identification of diatoms, cyanobacteria, and nematodes, respectively. Eduardo Luis Cupertino Ballester, Wilson Wasielesky Jr., Ronaldo O. Cavalli, and Paulo César Abreu are research fellows of CNPq (National Council for the Development of Science and Technology of Brazil), and Tito Luis Pisseti received a CNPq Master’s scholarship during the study.

References Abreu PC, Ballester ELC, Odebrecht C, Wasielesky W Jr, Cavalli RO, Granéli W, Anésio AM (2007) Importance of biofilm as food source for shrimp (Farfantepenaeus paulensis) evaluated by stable isotopes (d13C and d15N). J Exp Mar Biol Ecol 347(1-2):88–96. https://doi.org/10.1016/j.jembe.2007.03.012 Akiyama D, Dominy WG, Lawrence AL (1992) Penaeid shrimp nutrition. In: Fast AW, Lester LJ (eds) Marine shrimp culture: principles and practices. Elsevier, Amsterdam, pp 535–568. https://doi.org/10.1016/B978-0444-88606-4.50031-X Allan GF, Maguire GB (1992) Effects of stocking density on production of Penaeus monodon Fabricius in model farming ponds. Aquaculture 107(1):49–66. https://doi.org/10.1016/0044-8486(92)90049-Q Allan GF, Moriarty DJW, Maguire GB (1995) Effects of pond preparation and feeding rate on production of Penaeus monodon Fabricius, water quality, bacteria and benthos in model farming ponds. Aquaculture 130(4):329–349. https://doi.org/10.1016/0044-8486(94)00316-G Anderson RK, Parker PL, Lawrence AL (1987) A 13C/ 12C tracer study of the utilization of presented feed by commercially important shrimp Penaeus vannamei in a pond growout system. J World Aquacult Soc 18(3): 148–155. https://doi.org/10.1111/j.1749-7345.1987.tb00433.x Ballester ELC, Wasielesky W Jr, Cavalli RO, Abreu PC (2007) Nursery of the pink shrimp Farfantepenaeus paulensis in cages with artificial substrates: biofilm composition and shrimp performance. Aquaculture 265(1-4):355–362. https://doi.org/10.1016/j.aquaculture.2007.04.003 Bratvold D, Browdy CL (2001) Effects of sand sediment and vertical surfaces (AquaMats™) on production, water quality, and microbial ecology in an intensive Litopenaeus vannamei culture system. Aquaculture 195(1-2):81–94. https://doi.org/10.1016/S0044-8486(00)00538-X Costa RC, Lopes M, Castilho AL, Fransozo A, Simões SM (2008) Abundance and distribution of juvenile pink shrimps Farfantepenaeus spp. in a mangrove estuary and adjacent bay on the northern shore of São Paulo state, southeastern Brazil. Invertebr Reprod Dev 52(1-2):51–58. https://doi.org/10.1080 /07924259.2008.9652272 Cuzon G, Lawrence A, Gaxiola G, Guillaume J (2004) Nutrition of Litopenaeus vannamei reared in pond or in tanks. Aquaculture 235(1-4):513–551. https://doi.org/10.1016/j.aquaculture.2003.12.022 D'Incao F (1991) Pesca e biologia de Penaeus paulensis na Lagoa dos Patos, RS. Atlantica 13:159–169 [In Portuguese with English abstract] D’Incao F, Reis EG (2002) Community-based management and technical advice in Patos Lagoon estuary (Brazil). Ocean Coast Manag 45(8):531–539. https://doi.org/10.1016/S0964-5691(02)00084-4 Dutra FM, Moretto Y, Portz L, Ballester ELC (2016) Pen culture of Macrobrachium amazonicum: use of artificial diet and impact on benthic community. Aquac Res 47(1):266–275. https://doi.org/10.1111 /are.12488 EPP MA (2002) Stable isotopes in shrimp aquaculture. World Aquaculture 33:18–19 Fast AW, Lester LJ (1992) Marine shrimp culture: principles and practices. Elsevier, Amsterdam Jeffrey SW, Humphrey GF (1975) New espectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanz 167(2):191–194. https://doi. org/10.1016/S0015-3796(17)30778-3 Jensen L, Domingos JAS, Peixoto S, Santos MHS, Cavalli RO, Poersch LH, Ballester ELC, Wasielesky WJ (2016) Cultivo do camarão rosa em cercados: produção e viabilidade econômica. In: WJr W, Poersch LH (eds) Cultivo de camarões em gaiolas e cercados no estuário da Lagoa dos Patos. Editora da FURG, Rio Grande, pp 150–165

Aquacult Int Jensen LV, Wasielesky W Jr, Cavalli RO, Peixoto S, Santos MHS, Ballester ELC (2004) Growth and survival of pink shrimp (Farfantepenaeus paulensis) postlarvae in cages and pen enclosures. Sci Agric 61(3):332–335. https://doi.org/10.1590/S0103-90162004000300016 Jensen LV, Wasielesky W Jr, Ballester ELC, Cavalli RO, Santos MS (2006) Role of the microalgae Thalassiosira fluviatilis in weight gain and survival of the shrimp Farfantepenaeus paulensis reared in indoor nursery tanks. Nauplius 14:37–43 Komárek J, Anagnostidis K (2005) Cyanoprokaryota. In: Büdel B, Gärtner G, Krienitz L, Scagerl M (eds) Süsswasseflora von Mitteleuropa 19/2. Elsevier, Spektrum, Heidelberg, p 759 Krumenauer D, Cavalli RO, Ballester ELC, Wasielesky W Jr (2010) Feasibility of pacific white shrimp Litopenaeus vannamei culture in Southern Brazil: effects of stocking density and single or double crop management strategy in earthen ponds. Aquac Res 41(2):240–248. https://doi.org/10.1111/j.13652109.2009.02326.x Krummenauer D, Zogbi PR, Cavalli RO, Peixoto S, Wasielesky WJ (2016) Análise da viabilidade do cultivo do camarão rosa Farfantepenaeus paulensis em gaiolas em diferentes densidades de estocagem durante o outono no extremo sul do Brasil. In: Wasielesky W Jr, Poersch LH (eds) . Cultivo de camarões em gaiolas e cercados no estuário da Lagoa dos Patos. Editora da FURG, Rio Grande, pp 166–173 Kumlu M, Eroldogan OT, Saglatimur B (2001) The effects of salinity and added substrates on growth and survival of Metapenaeus monoceros (Decapoda: Penaeidae) post-larvae. Aquaculture 196(1-2):177–188. https://doi.org/10.1016/S0044-8486(00)00580-9 Moss KRK, Moss SM (2004) Effects of artificial substrate and stocking density on the nursery production of pacific white shrimp Litopenaeus vannamei. J World Aquacult Soc 35(4):537–542. https://doi.org/10.1111 /j.1749-7345.2004.tb00121.x Nunes AJP, Gesteira TCV, Goddard S (1997) Food ingestion and assimilation by the southern brown shrimp Penaeus subtilis under semi-intensive culture in NE Brazil. Aquaculture 149(1-2):121–136. https://doi. org/10.1016/S0044-8486(96)01433-0 Ostrensky A, Wasielesky W Jr (1995) Acute toxicity of ammonia to various life stages of the São Paulo shrimp, Penaeus paulensis Pérez-Farfante, 1967. Aquaculture 132(3-4):339–347. https://doi.org/10.1016/0044-8486 (94)00343-M Otoshi CA, Montgomery AD, Matsuda EM, Moss SM (2006) Effects of artificial substrate and water source on growth of juvenile pacific white shrimp, Litopenaeus vannamei. J World Aquacult Soc 37(2):210–213. https://doi.org/10.1111/j.1749-7345.2006.00029.x Peixoto S, Wasielesky W Jr, Louzada L (2003) Comparative analysis of pink shrimp, Farfantepenaeus paulensis, and Pacific White shrimp, Litopenaeus vannamei, culture in extreme southern Brazil. Journal of Applied Aquiculture 14(1-2):47–56. https://doi.org/10.1300/J028v14n01_07 Poersch L, Castello JP, Wasielesky W Jr, Cavalli RO (2007) The challenge of sustainable aquaculture: effects on the environment of the Patos Lagoon estuary. J Coast Res 47:130–135. https://doi.org/10.2112/1551-503647.sp1.130 Porter KG, Feig YS (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr 25(5):943–948. https://doi.org/10.4319/lo.1980.25.5.0943 Preto AL, Cavalli RO, Pissetti T, Abreu PC, Wasielesky W Jr (2005) Efeito da densidade de estocagem sobre o biofilme e o desempenho de pós-larvas do camarão-rosa Farfantepenaeus paulensis cultivadas em gaiolas. Ciência Rural 35(6):1417–1423. [In Portuguese with English abstract]. https://doi.org/10.1590/S010384782005000600030 Quaresma J, Sugai JK (2005) Circadian profile of feed consumption and amylase and maltase activities in the juvenile shrimp Farfantepenaeus paulensis. J World Aquaculture Soc 36(1):141–147. https://doi. org/10.1111/j.1749-7345.2005.tb00141.x Round FE, Crawford RM, Mann DV (1990) The diatoms: biology and morphology of the genera. Cambridge University Press, Cambridge Ruas VM, Dumont LF, D’Incao F (2011) Evaluation of the capture size and abundance of pink shrimp Farfantepenaeus paulensis (Pérez Farfante 1967) around the Marinheiros Island. Patos Lagoon Estuary, RS, Brazil, Atlântica 33:161–172 [In Portuguese with English abstract] Samocha TM, Lawrence AL, Biedenbach JM (1993) The effect of vertical netting and water circulation pattern on growth and survival of Penaeus vannamei postlarvae in an intensive raceway system. J Appl Aquiculture 2(1):55–64. https://doi.org/10.1300/J028v02n01_04 Schlechtriem C, Ricci M, Focken U, Becker K (2004) Mass produced nematodes Panagrellus redivivus as live food for rearing carp larvae: preliminary results. Aquac Res 35(6):547–551. https://doi.org/10.1111/j.13652109.2004.01049.x Silva CF, Ballester ELC, Monserrat J, Geracitano L, Wasielesky W Jr, Abreu PC (2008) Contribution of microorganisms to the biofilm nutritional quality: protein and lipid contents. Aquac Nutr 14(6):507–514. https://doi.org/10.1111/j.1365-2095.2007.00556.x

Aquacult Int Snedecor G, Cochran WG (1982) Statistical Methods, 7th ed. State University Press, Ames - Iowa, USA Soares R, Peixoto S, Bemvenuti C, Wasielesky W Jr, D’Incao F, Murcia N, Suita S (2004) Composition and abundance of invertebrate benthic fauna in Farfantepenaeus paulensis culture pens (Patos Lagoon estuary, Southern Brazil). Aquaculture 239(1-4):199–215. https://doi.org/10.1016/j.aquaculture.2004.05.041 Soares R, Peixoto S, Wasielesky W Jr, D’Incao F (2005) Feeding rhythms and diet of Farfantepenaeus paulensis under pen culturein Patos Lagoon estuary, Brazil. J Exp Mar Biol Ecol 322(2):167–176. https://doi. org/10.1016/j.jembe.2005.02.019 Sokal RR, Rohlf FJ (1969) Biometry: principle and practices of statistics in biological research. W. H. Freeman and Company, New York, USA Strickland JDH, Parsons TR (1972) A practical handbook of seawater analysis. Journal of the Fisheries Research Board of Canada, Ottawa Thompson FL, Abreu PC, Wasielesky W Jr (2002) Importance of biofilm for water quality and nourishment in intensive shrimp culture. Aquaculture 203(3-4):263–278. https://doi.org/10.1016/S0044-8486(01)00642-1 UNESCO - United Nations Educational, Scientific and Cultural Organization (1983) Manuals and guides 12: chemical methods for use in marine environmental monitoring, UNESCO, Paris, http://www.jodc.go. jp/info/ioc_doc/Manual/055950eo.pdf. Cited 10 nov 2016 Utermöhl H (1958) Zur Vervollkommnung der quantitativen Phytoplankton Methodik. Verhandlungen der Internationalen Vereinigung fur Theoretische und Angewandte Limnologi 9:1–38 Van Wyk P, Scarpa J (1999) Water quality requirements and management. In: Van Wyk P (ed) Farming marine shrimp in recirculating freshwater systems, Florida Department of Agriculture and Consumer Services. Tallahasee - Florida, USA, pp 128–138 Wasielesky W Jr, Abreu PC, Poersh LH, Thompson F, Ballester ELC (2011) Influence of light intensity on biofilm formation and the performance of pink shrimp Farfantepenaeus paulensis juveniles reared in cages. Aquac Res 43(5):706–712. https://doi.org/10.1111/j.1365-2109.2011.02878.x Wasielesky W Jr, Peixoto S, Jensen L, Poersch L, Bianchini A (2004) Estudo preliminar do cultivo do camarãorosa Farfantepenaeus paulensis em cercados no estuário da Lagoa dos Patos. Bol Inst Pesca 30:63–70 [In Portuguese with English abstract] Wasielesky W Jr, Peixoto S, Santos MHS, Cavalli RO (2002) Cultivo do camarão-rosa como alternativa de geração de renda. In: Alderón AI, Sampaio H (eds) Extensão universitária: ação comunitária em Universidades Brasileiras. Olho D'Água, São Paulo, Brasil, pp 17–27 Wasielesky W Jr, Poersh LH, Jensen L, Bianchini A (2001) Effect of stocking density on pen reared pink shrimp Farfantepenaeus paulensis (Pérez-Farfante, 1967) (Decapoda, Penaeidae). Nauplius 9:163–167 Zhukova NV, Kharlamenko VI (1999) Sources of essential fatty acids in the marine microbial loop. Aquat Microb Ecol 17:153–157. https://doi.org/10.3354/ame017153