Particle size preference, gut filling and evacuation rates of the rotifer Brachionus “Cayman” using polystyrene latex beads

Aquaculture 282 (2008) 75–82

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e

Particle size preference, gut filling and evacuation rates of the rotifer Brachionus “Cayman” using polystyrene latex beads Andreas Baer a,⁎, Chris Langdon c, Scott Mills d, Carsten Schulz a, Kristin Hamre b a

Institute of Animal Breeding and Husbandry, Christian Albrechts University, Kiel, Germany National Institute of Nutrition and Seafood Research, Bergen, Norway Department of Fisheries and Wildlife, Coastal Oregon Marine Experiment Station, Oregon State University, Newport, OR, USA d The Academy of Natural Sciences, 1900 Benjamin Franklin Parkway Philadelphia, PA, USA b c

A R T I C L E

I N F O

Article history: Received 27 February 2008 Received in revised form 10 June 2008 Accepted 11 June 2008 Keywords: Brachionus plicatilis Brachionus “Cayman” Selective feeding Particle size Gut filling time Gut evacuation time

A B S T R A C T A rotifer strain commonly used in Norwegian cod hatcheries was identified as Brachionus “Cayman” using genetic barcoding techniques. This rotifer is used extensively in hatcheries around the globe being a member of the Brachionus plicatilis species complex. It is of medium lorica size (168 ± 10 × 111 ± 9 × 81 ± 8 μm3) and is most closely related to B. “Tiscar”, its closest named relative being B. ibericus. Size selective feeding by Brachionus “Cayman” was investigated using polystyrene latex beads with diameters of 1.6 to 20 μm. Different sizes of latex beads were offered to the rotifers, in combination with a standard 6 μm bead, and Jacobs selectivity indices (D) were calculated based on the relative numbers of the different sized beads consumed. In addition, experiments were carried out to determine the optimal feeding period for short-term enrichment of rotifers in order to maximize the volume of ingested beads. Gut evacuation rates at different water temperatures (4, 10 and 26 °C) were also determined. B. “Cayman” showed the highest positive selectivity (D = +0.54) for 4.5 μm latex beads compared to other beads in the size range 1.6 to 20 μm. The maximum diameter of latex bead ingested was 10 μm, while 12 and 15 μm beads were captured but not ingested. Rotifers ingested a mean bead volume of 18,000 μm3 (S.D. + 2051) after 35 min of feeding on 4.5 μm diameter beads at a volumetric concentration of 40 × 107 μm3 mL− 1 at 26 °C. This corresponds to approximately 1.18% of the volume of the whole body of the rotifer. Rotifers emptied their guts at slower rates when culture temperatures were lowered from 26 to 4 °C. These results will facilitate optimization of enrichment protocols for rotifers in order to improve their nutritional value as live feed for marine fish larvae. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Rotifers are commonly used as a live feed for the early larval stages of marine fish larvae (Lubzens et al., 1989). Due to possible nutritional deficiencies, rotifers are often fed on supplements to improve their nutritional value (Hamre, 2006). Widely used rotifer diets include microalgae (Nannochloropsis and Tetraselmis spp.) and yeast (Dhert et al., 2001; Srivastava et al., 2006). Because of the variable nutritional value of such natural diets, researchers have attempted to further enrich rotifers with lipid emulsions and artificial microparticulate diets (Kolkovski, 2001; Önal & Langdon, 2000; Tucker, 1998; Kumlu & Jones, 1995; Yúfera et al., 1995). It is important to know particle size preferences for ingestion by rotifers in order to prepare enrichment particles in the optimal size range. Previous feeding experiments with Brachionus spp. rotifers fed on mixtures of algal species of different cell diameters showed that the preferred food particle size differed among rotifer species (Rothhaupt, ⁎ Corresponding author. Tel.: +49 431 880 4363; fax: +49 431 880 2588. E-mail address: [email protected] (A. Baer). 0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.06.020

1990; Vadstein et al., 1993). In this study, we avoided using different algae species in particle size selection experiments and, instead, used different sizes of latex–polystyrene beads. The main advantage of using a single type of inert bead to study particle size selection is that it's possible to avoid the effects of particle surface characteristics, feeding attractants and nutritional quality on size selection measurements. In this study we examined the preferred particle size for ingestion for the rotifer Brachionus “Cayman” as well as gut filling and evacuation rates — the latter under a range of temperatures to simulate commercial hatchery protocols for enriching rotifers. Because of prevailing confusion surrounding the taxonomy of Brachionus sp. rotifers used in aquaculture, the rotifer strain used in this study was identified with the aid of genetic barcoding techniques. The examined strain was originally imported to Norway from a Spanish hatchery in 1998 by Joachim Stoss, Stolt Sea Farm's turbot hatchery, Øye. Since then, it has been supplied to several cod hatcheries in Europe. Stable cultures of this strain can be cultured in full (30–35 ppt) seawater (Stoss, personal communication). Recent genetic analysis has indicated that Brachionus plicatilis is not a single species but is, in fact, a complex of at least 15 putative

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species (Ciros-Perez et al., 2001; Gomez et al., 2002; Suatoni et al., 2006). Identifying the species used in experiments or hatcheries is a critical step toward a more scientific approach to understanding rotifer biology. To elaborate, B. plicatilis was the subject of approximately 750 peer-reviewed papers between 1950 and 2000 (Yúfera, 2001). Unfortunately, for the majority of these articles, we do not know which of the 15 putative species were used thus limiting the utility of this large accumulation of knowledge. This is especially important, considering that Brachionus sp., Kartella sp. and Synchatea sp. rotifers display different size preferences for food particles (Rothhaupt, 1990; Ronneberger, 1998), and within the B. plicatilis species complex, inter-specific differences in trophi size may also affect particle size selection (Ciros-Pérez et al., 2001). In order to clarify the identity of our experimental strain we chose to amplify and analyse a 712 base pair fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI or cox1). COI, often termed

the barcoding gene, exhibits the strongest capacity to identify species of any mitochondrial animal gene tested to date (Hebert et al., 2003). Unfortunately, COI suffers from a relatively rapid saturation of substitution sites such that while it is good for identifying species it tends to perform poorly when reconstructing phylogenetic relationships between more distantly related species. Due to the shortcomings of COI in phylogenetic reconstruction we chose to identify the strains position within the B. plicatilis species complex by examining nuclear ribosomal internal transcribed spacer 1 (ITS1) sequences deposited in GenBank, covering all the known members of the B. plicatilis species complex. The objectives of this paper were to measure a) size selectivity for food particles, b) gut filling rate and c) gut evacuation rate in a rotifer species commonly used in marine aquaculture. Genetic analyses were carried out to identify the experimental rotifer species but a detailed morphological description was outside the scope of this study.

Fig. 1. ITS1 and COI phylogenetic reconstructions. The ITS1 phylogenetic tree contains representative sequences from all members of the Brachionus plicatilis species complex currently deposited in GenBank, accession numbers are given at the leaves of the tree. Species names in bold text indicate currently accepted taxons under the International Code of Zoological Nomenclature, all other species are yet to be named and characterised, including the strain used in this study; B. “Cayman”. Each shaded region in the ITS1 phylogeny represents a genetic clade, the topmost shaded areas concur with the designation of L, SM and SS/S strains of the B. plicatilis species complex as used in aquaculture. All branches with less than 50% bootstrap support were collapsed. The locations from which the specimens in the COI phylogeny were sourced are indicated in the inset map by symbols, individuals that are also sequenced for ITS1 are indicated in a similar fashion. Symbols in red indicate that the specimens were sourced from Laboratories or Aquaculture facilities, while those in black are of a more definitive “wild” geographic origin. The test specimen is indicated in yellow highlighting on the COI phylogeny. Bootstrap support and posterior support for the maximum likelihood and Bayesian analyses are given at nodes (ML/Bayesian) for both the ITS1 and COI phylogenies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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2. Materials and methods 2.1. Species identification 2.1.1. DNA extraction, PCR amplification and genetic sequencing A clonal population of B. “Cayman” was established, at the Academy of Natural Sciences in Philadelphia, from a single amictic female and cultured in 16 ppt Instant Ocean® (Spectrum Brands) mixed with distilled water at a temperature of 20 °C under constant illumination. Five of the clonal, egg bearing, amictic females were washed in a Petri dish containing distilled water for 1 min. The washed individuals were then placed in a sterile 200 μL centrifuge tube containing 35 μL of InstaGene™ Matrix (Bio-Rad Laboratories) at 4 °C and subsequently heated to 56 °C for 20 min, 100 °C for 10 min, and then cooled at 4 °C for 30 min, the DNA extraction was stored at 4 °C until needed. The COI PCR primers LCO1490 and HCO2198 (Folmer et al., 1994) were used to amplify a 712 bp fragment of the COI gene from the DNA extract. A reaction volume of 20 μL containing 18 μL of 1.1× Taq complete (Gene Choice®), 0.25 μM of each primer and 1 μL of the extracted DNA template was used for the PCR reaction. The sample was subject to a thermocycling regime with an initial denaturing cycle of 3 min at 93 °C, followed by 40 cycles of 15 s at 92 °C, 20 s at 50 °C and 1 min at 70 °C, with a final incubation of 3 min at 72 °C. PCR samples were then shipped to Macrogen (Korea) for both forward and reverse sequencing. The resulting sequence was deposited in GenBank [Accession number EU289219]. 2.1.2. Phylogenetic reconstruction An additional 11 COI sequences for B. “Cayman” were downloaded from GenBank, along with 30 ITS1 sequences with unique haplotypes that were indicated as belonging to the species complex from previous studies (Gomez et al., 2002; Suatoni et al., 2006). In order to root the ITS1 phylogenetic reconstruction, three freshwater Brachionus sp. were also included in the analysis as outgroup taxa. GenBank accession numbers for all of the additional COI and ITS1 sequences used in the phylogenetic analyses are indicated in Fig. 1. As COI sequences contained no indels, alignment was made by eye using BioEdit v7.0.4 (Hall, 1999). In contrast, ITS1 sequences contain many indels; hence a rigorous regime of sequence alignment was pursued. A multiple alignment for ITS1 using the default parameters of ClustalX was constructed (Thompson et al., 1997), followed by manual fine tuning to obtain the most parsimony alignment with the aid of Tune ClustalX (Hall, 2004). A maximum likelihood (ML) phylogenetic reconstruction was implemented in PHYML (Guindon and Gasquel, 2003), for both ITS1 and COI datasets, using the model of nucleotide substitution estimated from log-likelihood parameters following the Akaike information criterion in ModelGenerator (Keane et al., 2006) with topological support estimated from 1000 bootstrap pseudoreplicates. In addition, a Bayesian analysis was conducted to obtain a phylogenetic reconstruction with posterior probabilities for nodes using 600,000 and 200,000 Markov chain Monte Carlo generations, for the ITS1 and COI DNA fragments respectively, in MrBayes 3.1.1 (Huelsenbeck and Ronquist 2001). Summaries of this analysis were derived with a burnin value determined by the asymptotic convergence of two independent runs as noted by the average standard deviation of the split frequencies sampled every 100 generations. 2.2. Experimental methods 2.2.1. Rotifer cultures The rotifers were obtained from Cod Culture Norway AS, Øygarden, who had obtained their rotifers from Stolt Sea Farm, Øye. At NIFES, the rotifers were reared in an aerated 20 L seawater tank at a salinity of 34 (ppt), a temperature of 26 °C, illuminated with dim light and at low

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density of 20 rotifers mL − 1 to maintain cultures in a healthy state. Fifty percent of the culture water was exchanged three times per week. A marine algal paste [Instant Algae 3600 (Nannochloropsis and Tetraselmis spp.); Reed Mariculture Inc., USA] was added to the cultures at each water change as a rotifer feed. 2.2.2. General experimental methods Rotifers were pre-starved for one day prior to each feeding experiment and then collected on a 60 μm mesh sieve, rinsed and re-suspended in GF/C (Whatman) filtered seawater. Aliquots of the resuspended rotifers were distributed into 10 mL plastic screw-capped test-tubes at densities of between 60 and 270 rotifers mL− 1 and fed on polystyrene latex beads. One test-tube without added rotifers served as a control for losses of beads in each trial. During the feeding period, the test-tubes were agitated on a shaker table to ensure oxygenation and to prevent bead sedimentation. After a feeding period of 10 min, rotifers were gently washed on a 60 μm mesh sieve to remove uneaten beads and beads sticking to the surface of the rotifers, then fixed with formalin (4%v/v; buffered to pH 8.00) and examined under an epifluorescence microscope to determine the number of ingested beads of each size class. Due to the fact that we found 12–20 μm beads inside the mastax, it is unlikely that gentle washing caused loss of gut contents before fixation. If there were losses of gut contents, it would be unlikely that there was differential loss of different sizes of beads that would cause an error in determination of selection indices. In the gut filling and evacuation experiments, rotifers were fixed immediately after sampling and washed later due to handling time constraints. A range of sizes (1.6 to 20 μm) of carboxylated polystyrene latex microspheres were supplied as monodispersed suspensions (Fluoresbrite®; Polysciences Inc., USA). All bead sizes were fluorescent (441 nm excitation; 486 nm emission) to facilitate microscopic enumeration, except for 12 and 15 μm-sized beads which were large enough to be easily counted using only transmitted light. 2.3. Experiment 1: particle size selection We used Jacob's index to determine particle size selection by comparing ingestion of equal volumes of each test bead size against a standard 6-μm bead size. Beads with nine different spherical diameters (SD) were tested: 1.6 μm, 2 μm, 3 μm, 4.5 μm, 6 μm, 10 μm, 12 μm, 15 μm and 20 μm (coefficient of variance 10%). Comparing ingestion of only two bead sizes rather than a mixture of many bead sizes resulted in higher potential rates of ingestion of large beads, increasing the accuracy of the estimated selection index. This approach was especially useful when estimating selection for bead sizes N6 μm that were ingested at low or apparent zero rates. Jacobs (1974) selectivity index (D) was calculated for two particle sizes, A and B, as: DA ¼ ðrA −pA Þ=ðrA þ pA −2rA pA Þ where rA = fA / (fA + fB) and pA = NA / (NA + NB) rA pA fA fB NA NB DA

fraction of latex beads of size A in the guts of the rotifers fraction of latex beads of size A in the medium number of latex beads of size A in the guts of the rotifers number of latex beads of size B in the guts of the rotifers number of latex beads of size A in the medium number of latex beads of size B in the medium selectivity for A ranges from −1 to +1, where D = 0 indicates unselective feeding and D = +1 indicates positive selection and D = −1 indicates negative selection for beads of size A.

A constant total bead volume of 5 × 106 μm3 mL− 1 for each bead size was provided to rotifers in each experiment in order to avoid

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confounding bead size with total offered bead volume. Bead concentrations were measured with a haemocytometer (Improved Neubauer, Labor Optic, Friedrichsdorf, Germany). A sample of the culture medium was taken immediately after addition of beads at the beginning of the experiment in order to ascertain the initial ratio of suspended bead sizes. Rotifers were fed on beads for 10 min, then between 30 and 80 rotifers were randomly sampled and the numbers of ingested test and standard 6 μm bead sizes determined using an epifluorescence microscope. To count ingested beads, formalin-fixed rotifers were transferred to a slide by pipette and carefully crushed under a cover slip until the ingested beads formed a single layer and could be easily counted. Samples from the control test-tube (without rotifers) were used to determine changes in bead size ratios after 10 min due to sedimentation or other losses. Three rotifer feeding trials, with triplicate test-tubes (3 × 3 test-tubes) per bead size, were set up for 2 μm and 3 μm sized beads while one trial (1 × 3 test testtubes) was set up for other bead sizes.

2.4.4. Statistics The software Statistica ver. 7 (Statsoft Inc., Tulsa, OK, U.S.) was used for statistical analyses and differences were considered significant at p b 0.05. Data used for parametric ANOVA were tested for normal distribution and homogeneity of variances (f-test, p b 0.05). In Experiment 1, Students t-tests (p b 0.05) were used to compare the volumes of ingested test beads with 6 μm-sized beads and Jacob's selection indices (D values) against a D value of zero. Total ingested volumes for different sizes of beads after 10 min of feeding were compared by one-way ANOVA followed by Tukey's HSD test. The nonparametric Kolmogorow–Smirnow test was used to compare the percent ingested volumes of test beads compared to the total ingested volume for mixtures of test beads and the standard 6-μm bead. Gut filling and evacuation rates were analysed by repeated measures ANOVA and Tukey's HSD test to determine significant differences in bead concentration among different sampling times within treatments. Student t-tests were used to compare differences between treatments at individual sampling times.

2.4. Experiment 2: gut filling and evacuation rates 3. Results 2.4.1. Gut filling rates Beads of the preferred size (4.5 μm; determined in Experiment 1) were fed to rotifers to determine gut filling and evacuation rates. To simulate typical conditions that cod hatcheries use to enrich rotifers, bead concentration was increased to provide a total volumetric concentration of 44 × 107 μm3 beads mL− 1. Only half of this volume was offered in another test test-tube in order to determine the effect of volumetric concentration on filling rate. From 250 to 500 rotifers mL− 1 were transferred to triplicate sets of 5 mL test test-tubes, with one triplicate set per sample time period. Beads were added and the cultures agitated on a shaker table. Rotifer samples were taken 3, 5, 10, 17, 25, 35, 45, 60, 80 and 120 min after initiation of feeding. The average number of beads ingested per rotifer was determined for each sampling period, according to methods described for Experiment 1. 2.4.2. Gut evacuation rates Rotifers were transferred to triplicate 5 mL test-tubes at a density of 500 rotifers mL− 1 and fed on 4.5 μm beads at 26 °C until they filled their guts (determined as described above). They were then gently sieved, washed and transferred to containers filled with bead-free seawater. Three culture temperatures (4, 10 and 26 °C) were tested to determine the effect of temperature on evacuation rates of rotifers. These temperatures were chosen to simulate rotifer-handling conditions in a typical cod hatchery where rotifers are typically enriched at 24–26 °C and stored at 4–6 °C before being fed to cod larvae at 10–12 °C. Rotifers were initially transferred to 5 mL plastic screw-capped test-tubes filled with 26 °C filtered (bead-free) seawater. The testtubes were then placed in 1.5 L plastic bottles filled with water at the desired final experimental temperature (4, 10 or 26 °C) to allow rotifers to gradually thermally adjust. After 10–15 min, the water temperature in the test-tubes reached the desired experimental temperatures and the rotifers were transferred from the test-tubes to the 1.5 L bottles. The 1.5 L bottles were held in water-baths in order to maintain water temperatures. Rotifer samples were taken from the 1.5 L plastic bottles over a time interval from 30 min to 18 h of starvation (Fig. 7). To avoid stress during sampling, rotifers were gently siphoned from the bottles and fixed immediately in formalin (4%v/v). Beads inside the rotifers were counted using an epifluorescence microscope. 2.4.3. Rotifer size Rotifers were sampled and photographed under a microscope (Olympus BX 51) at 100× magnification, while swimming freely in a drop of seawater on a microscope slide. Measures of length, width and depth of 25 rotifers were taken using Olympus DP-soft and volumes calculated assuming the rotifer was cube shaped.

3.1. Species identification The ITS1 alignment contained 30 haplotypes and was 351 bases long due to the inclusion of a number of indels, with individual sequences ranging from 314 to 331 bases in length. The COI alignment contained 12 unique haplotypes with a length of 603 bases. The model of DNA evolution that best described the ITS1 data was the Kimura 3 parameter; variable base frequencies, equal transition frequencies, variable transversion frequencies with a gamma distributed rate variation among sites (K81uf+G). The COI data set was also characterised by this model but with a proportion of invariable sites and no gamma distributed rate variation among sites (K81uf+I). The phylogenetic reconstruction indicated 5 major genetic clades composed of 15 separate species. Of these clades the most species rich, containing 40% of the putative species, is the one that conforms to the SM strain of rotifers as used in aquaculture, to which B. “Cayman” belongs. Many of these species have not yet been named and are only identified by their type localities from previous genetic studies (Fig. 1; Gómez 2002 et al., Suatoni et al., 2006). The subject of the current study was identified as B. “Cayman”, a name assigned in the first major genetic characterisation of the species complex by Gómez et al., (2002). As yet this species has not been formally classified under the zoological taxonomic code and hence is only defined by the locality from which it was first sequenced Grand “Cayman” Island, Meagher Pond. Examination of the ITS1 phylogeny suggests that B. “Cayman” is most closely related to B. “Tiscar”, with the only named species in this group being B. ibericus, see Fig. 1. The group also includes two unnamed species found on the Australian continent B. “Coyrecupiensis” and B. “Towerinniensis”, also of medium size range, and B. “Almenara” known from sites in Spain and the United States of America (Gomez et al., 2002, Suatoni et al., 2006). B. “Cayman” has also been isolated from the east coast of the USA and Turkey (Gomez et al., 2002; Suatoni et al., 2006), with the COI phylogeny hinting at a potential cosmopolitan distribution for B. “Cayman” (see inset map Fig. 1). 3.2. Preferred particle size The ratios of 1 and 3 μm beads to 6 μm standard beads suspended in water were similar to the ratio of beads inside the rotifer; however, rotifers selectively ingested 4.5 μm-sized beads but selected against 2 and 10 μm beads (Fig. 2). Except for 10 μm-beads, the total ingested volumes for the mixtures of bead sizes (6 μm + tested bead) were comparable and ranged between 2989 and 3455 μm3 (average 3402 μm3) per rotifer over a 10 min feeding period (Fig. 3). Beads that were 12, 15 or 20 μm in size were not ingested by rotifers;

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Fig. 2. Ratios (number of tested beads/number of 6 μm beads) of beads suspended in water and ingested by rotifers. Each bead size was offered in combination with 6 μm sized latex beads at identical volumes (5 × 106 μm3 mL− 1). The ratios of 10 to 6 μm-sized beads suspended in water and in the rotifers were 0.27 and 0.18, respectively. All values are given as means + S.D. and n = 26–84. Error bars represent standard deviations. Letters denote significant differences between suspended and ingested bead ratios (Mann–Whitney-U-Test, t-test, p b 0.05).

therefore, ingested total bead volumes for mixtures with these bead sizes are represented by ingestion of 6 μm beads alone. The fraction of test beads making up the total ingested volume increased from 44% for 1 μm- sized to 72% for 4.5 μm-sized beads (Fig. 4); however, 10 μmsized beads represented only 39% of the total ingested volume even though the rotifers ingested the greatest total volume of beads when fed on a mixture of 10 μm and 6 μm beads. As mentioned above, 12 μmsized and 15 μm-sized beads were captured but not ingested by rotifers (Fig. 3; stippled bars); therefore, 6-μm beads represented 100% of the volume of ingested beads when rotifers were fed on these bead mixtures (12, 15 and 20μm-sized beads). Of the tested sizes of beads, rotifers showed strongest preference for 4.5 μm beads (Fig. 5, selectivity index D = 0.54, significantly different from zero; t-test, p b 0.05). The D values for all other bead sizes were not significantly different from zero. No selectivity index was calculated for 12, 15 and 20 μm-sized beads because these beads were not ingested by rotifers. 3.3. Gut filling rates Maximum gut fullness with 4.5 μm beads was reached after 35 min of feeding with an average of 350 beads (17848 μm3) retained by each rotifer exposed to bead densities of 40× 107 μm3 mL− 1 (Fig. 6). After 35 min, the average number of retained beads declined significantly (ttest, p b 0.05) to 200 and 235 beads at 80 and 120 min of feeding, respectively. The gut filling rate curve was similar in shape when offering only 20× 107 μm3 mL− 1 of beads, reaching a maximum at 233 beads per

Fig. 3. Ingested total combined volumes for each bead size with the 6 μm standard bead after 10 min feeding. All values are means + S.D.; n = 26–84. Note that 12 μm-, 15 μm- and 20 μm beads were not ingested and the bars for these bead sizes represent ingestion of only the 6 μm-sized beads. Letters denote significant differences between the bead sizes, ANOVA, Tukey (p b 0.05).

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Fig. 4. Total volumes of each ingested bead size expressed as a percent of the total combined ingested volume for the test bead size and the 6 μm standard bead fed to rotifers. Percent values are means + S.D., n = 26–84. Letters denote significant differences (p b 0.05) among tested bead sizes, ANOVA, Kolmogorow–Smirnow test (p b 0.05) was used to compare the percent ingested volumes of test beads to the total ingested volume for mixtures of test beads and the standard 6-μm bead.

rotifer after 34 min of feeding and remaining at this level for an additional 20 min. The average number of beads per rotifer decreased (but not significantly, Tukey's test, p N 0.05) to 190 beads after 90 min. 3.4. Gut evacuation rates Rotifers showed the fastest gut evacuation rate at 26 °C (Fig. 7). After 120 min of starvation, rotifers had lost over 95% of ingested beads. At 10 °C, the rate of bead loss was high during the first 30 min of starvation but after 120 min, 40% of the beads remained and 18% still remained after 18 h. The slowest evacuation rate occurred when rotifers were transferred to 4 °C seawater. The loss of beads during the first hour of starvation was 31% of ingested beads, but evacuation rates were close to zero after 60 min. No significant difference in retained bead concentrations occurred between 60 min and 18 h of starvation. The length, width and depth of the rotifers were 168 ± 10, 111 ± 9 and 81 ± 8 μm, respectively, resulting in an estimated average rotifer volume of 1.51 × 106 μm3, assuming a cube-shaped rotifer. The average estimated maximum bead volume in the rotifer gut of 17,848 μm3 is, therefore, only 1.18% of the estimated volume of the rotifer's body. 4. Discussion 4.1. Species identification Genetic analysis of our test specimen indicates that it is B. “Cayman” belonging to the B. plicatilis species complex. It is related to a group of medium sized rotifers known in aquaculture as SM strains that form an independent genetic clade, based on an ITS1 phylogenetic reconstruction. It has been shown in previous genetic studies that this species is

Fig. 5. Selectivity index D (Jacobs 1974) for rotifers fed on standard-sized 6 μm beads in combination with other test bead sizes. D values express selectivities for 1.6 to 10 μm latex beads compared with the 6 μm standard bead size. Positive values express positive selection for the tested bead size. 4.5 μm-sized beads are significantly preferred compared with 6 μm beads (t-test, p b 0.05) but no significant selectivities were observed for the other bead sizes.

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Fig. 6. Retention of 4.5 μm beads by rotifers at volumetric particle concentrations of 40 × 107 μm3 beads mL− 1 — particle concentrations typically used to enrich rotifers in marine fish hatcheries. Gray points represent number of beads retained at 50% of this bead concentration (20 × 107 μm3 mL− 1). All values are means + SD, n = 3. Letters indicate significant difference within treatment (time). Repeated measures, ANOVA, Tukey's test (p b 0.05).

used extensively in marine finfish hatcheries throughout the world (Papakostas et al., 2006a, Papakostas et al., 2006b), making our findings widely applicable to numerous hatcheries, farming a variety of fish species. While it appears that B. “Cayman” may have a cosmopolitan distribution, many of the true collection localities for specimens used in previous genetic studies are unknown. Unfortunately, only a laboratory of origin is provided in the literature for many of the COI haplotypes downloaded from GenBank and included in our analysis. Further investigation is required to clarify this point, as members belonging to this putative species may have different physiological and morphological characteristics that might be of utility to aquaculture. For the haplotypes for which we have definitive geographic information, there is a clear pattern of isolation by distance, as noted for another member of the B. plicatilis species complex (Mills et al., 2007). A major division between North American individuals and the Middle Eastern representative is evident, with our specimen most closely related to the Turkish specimen. This finding is not surprising considering our strain was originally sourced from a Spanish Hatchery and suggests that our test specimen is most likely of European origin.

Unfortunately, polystyrene latex beads do not simulate some of the characteristics of natural foods that might be important in affecting ingestion and selection. For example, Rothhaupt (1990) found that B. angularis did not ingest 12 μm beads; however, he found that this rotifer species ingested 12 μm-sized algal cells (Chlamydomonas sphaeroides), perhaps because of the more flexible walls of algal cells compared with the harder beads (Hotos, 2003). Similarly, it is possible that our studied rotifers could ingest particles greater than 10 μm providing they are soft-walled and malleable. Furthermore, Hotos (2003) fed two different algae species (Chlorella sp. and Asteromonas gracilis) to an L and S strain members of the B. plicatilis species complex and found that rotifers ingested and preferred A. gracilis (16– 22 μm in cell size) compared with the smaller celled Chlorella sp. (2– 5 μm). Hotos (2003) suggested that flagellae of A. gracilis resulted in more efficient cell capture and ingestion. We found that 12 μm beads were captured but not ingested by B. “Cayman”. The upper ingestible particle size has been reported to be dependent on rotifer body size (Hino and Hirano, 1980) with larger rotifers being able to ingest larger particle sizes. It is likely that the size of the feeding structure (the mastax) and mouth opening influence the maximum particle size ingested by rotifers. Fontaneto and Melone (2006) reported that while gross morphology of the mastax of an Lstrain member of the B. plicatilis species complex did not vary among individuals, variation in mastax size was evident among individuals. Therefore, it is possible that variation in mastax size among individual rotifers within a population results in variation in the preferred size of ingested particles, as suggested by the large variation in the ratios of bead sizes ingested by rotifers observed in this study (Fig. 2). We found that B. “Cayman” showed similar volumetric filling rates over a 10 min feeding period that were not dependent on bead size over the size range of 1.6 to 6 μm (Fig. 3). A similar volumetric bead concentration was presented to rotifers for each bead size, suggesting that rotifers' clearance rates (ml mL of medium filtered per min) were constant for 1.6 to 6 μm beads. Consequently, rotifers captured, processed and ingested smaller individual beads at higher rates than larger beads in order to maintain constant volumetric ingestion rates. 4.3. Gut filling rates Rotifers in this study showed a rapid rate of gut filling over an initial 35 min feeding period with an ingestion rate of twelve 4.5 μm beads min− 1 (612 m3μm3 min− 1; Table 1) after 10 min of feeding at a bead concentration of 40 × 107 m3 μm3 mL− 1. Pre-starving rotifers before the beginning of the feeding experiment and use of beads rather than

4.2. Preferred particle size The rotifer species Brachionus “Cayman” (Gomez et al., 2002) examined in this study showed a significant preference for 4.5 μmsized polystyrene latex beads compared with other bead sizes from 1.6 to 20 μm in diameter, with an upper ingested particle size limit within the range of 10 to 12 μm. Previous investigations have shown that ingestion rate is dependent on particle size in the genus Brachionus (Chotiyaputta et al., 1978; Rothhaupt, 1990; Hlawa et al., 1994; Heerkloss et al., 1995; Hansen et al., 1997) and in freshwater zooplankton (Bern, 1990). Most of the examined rotifer species (B. angularis, B. calyciflorus, B. rubens) show a narrow range of ingestible particle sizes. Hansen et al. (1997) determined that the preferred feed particle size for an unidentified member of the B. plicatilis species complex was 8.3 μm, using different algal species that ranged in cell size from 1.4 to 21 μm; however, the possible effects of surface characteristics and “taste” of the different algal species on particle selection may have confounded the results (DeMott 1986). The advantage of using artificial beads is that the effects of other factors, apart from size, on particle selection can be eliminated.

Fig. 7. Rate of gut evacuation of bead-fed rotifers that were subsequently starved at different water temperatures. Values are means + S.D. Letters indicate significant differences within temperature treatments. Repeated measures ANOVA and Tukey's test (p b 0.05).

A. Baer et al. / Aquaculture 282 (2008) 75–82 Table 1 Filling rates shown as ingested beads per min for both feeding experiments Feeding time (min)

Bead size (μm)

Filling rate (beads/min)

Ingested bead volume/min (μm3)

Experiment 1 (volumetric concentration: 10 × 106 μm3 beads mL− 1) 10 1.6 59 10 2 15 10 3 12 10 4.5 4.5 10 6 2 10 10 0.4

144 81 180 229 222 211

Experiment 2 (Volumetric concentration: 44 × 107 μm3 beads mL− 1) 3 4.5 12 5 4.5 13 10 4.5 12 17 4.5 11 25 4.5 11 35 4.5 10 45 4.5 7 60 4.5 4 80 4.5 3 120 4.5 2

612 663 612 561 561 510 357 204 153 102

Table ‘Experiment 1’ shows the ingested numbers of test beads during the 10 min feeding trial. Because the test beads were offered together with 6-μm beads in each feeding trial the number of ingested 6-μm sized beads is an average throughout all performed experiments.

natural food particles may have affected initial feeding rates. Such rapid responses to food availability by rotifers may be useful for aquaculturists who want to rapidly “boost” the nutritional value of rotifers by feeding them on enrichment diets or supplements. There have been several studies on the optimal duration for short-term nutritional enrichment or “boosting” of rotifers with the objective of completely filling the guts of rotifers with dietary particles in a short period of time (see review by Lubzens et al., 1989). Longer-term enrichment results in greater absorption and assimilation of ingested nutrients; for example, Walford and Lam (1987) found that highest assimilation of supplemented n−3 HUFAs occurred after 12 h enrichment with microcapsules and Watanabe (1993) recommended an optimal enrichment period of 12 h using lipid emulsions. Upon commencement of feeding, rotifers filled their guts to reach a maximum volume of 18,000 μm3 after 35 min of feeding. After this time, the number of ingested beads gradually declined over the period from 35 to 80 min of feeding and then appeared to stabilize after 120 min at about 12,000 μm3 (Fig. 5). Reducing initial bead concentrations to 20 × 107 μm3 mL− 1 resulted in lower total gut volumes after 34 min of feeding, suggesting that ingestion rates may have been limited by available volumetric food concentrations. Kleinow et al., (1991) estimated, by means of transmission electron microscopy, that the volume of the stomach and intestine of an L-strain member of the B. plicatilis species complex was between 60 and 120 plpL. In comparison, we found that the maximum volume of ingested beads to be about 18,000 μm3 or 18 pl pL after 35 min of feeding. This difference in these estimated gut volumes could be due to the larger size of the B. plicatilis strain observed by Kleinow et al., (1991) compared with B. “Cayman”. Secondly, we did not take into account the volume occupied by spaces between ingested beads that would result in an underestimate of gut volume and thirdly, it is possible that ingested beads only filled part of the digestive system. The estimated small proportion of the whole rotifer body volume (excluding the egg sacs) occupied by the gut (1.18% v/v) was also unexpected based on observations of bead-fed rotifers under the microscope; however, failure to take into account the three-dimensional aspect of the rotifer's gut and body could explain why these perceptions may be misleading. If the gut volume occupied by ingested food is only about 1% of the total rotifer body volume, short-term “boosting” the nutritional composition of rotifers by filling their guts with dietary particles might not be very effective and longer-term enrichment may be necessary to ensure

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greater changes in nutritional composition via uptake of nutrients into the rotifer's tissues. Clearly, additional studies need to be conducted to obtain more accurate estimates of the volume of the rotifer's body that is capable of being filled with ingested enrichment supplements. 4.4. Gut evacuation rates Rapid losses of ingested nutrients can occur if they are defecated or metabolized by rotifers (Helfman et al., 1997). Dhert (1996) has reported that rotifers evacuate their guts after 20–30 min at 25 °C. In this study, we found that gut evacuation rates were dependent on water temperature, with higher temperatures resulting in faster losses of beads. Montagnes et al. (2001) described the effects of temperature on grazing rates of B. plicatilis in terms of a Q10 relationship and, with additional data, the same approach could be used to describe the effects of temperature on gut filling and evacuation rates (Cossins and Bowler, 1987). In this study, we gradually cooled cultures to 4 °C to avoid cold-shocking the rotifers, resulting in possible rapid evacuation of gut contents (Børre Erstad, personal communication); however, this strategy still resulted in a 31% loss of beads over the initial cooling period (Fig. 6). Losses ceased after about 1 h of cooling to 4 °C and about 60% of ingested beads remained after 18 h of starvation. Concentrations of defecated beads in the 1.5 L culture were low (b102 beads mL− 1) and re-ingestion of defecated beads was considered to be insufficient to significantly refill the rotifers' guts. These results indicated that the practice of commercial hatcheries of cooling enriched rotifers before feeding them to fish larvae should result in retention of most of the ingested nutrients; however, further experiments should be conducted to investigate the effect of water temperature on rotifer swimming activity and survival as cold shock or low temperatures or both may cause rotifers to cease swimming and settle out of suspension, negatively affecting their availability for fish larvae. 5. Conclusions This study addressed factors affecting short-term enrichment or “boosting” of the rotifer B. “Cayman” to improve its nutritional values for fish larvae. Unlike many previous studies on members of the B. plicatilis species complex, our use of genetic markers allowed results on feeding physiology to be related to a taxonomically identified rotifer type. We found that rotifers preferentially ingested beads 4.5 μm in diameter compared to other bead over a size range of 1.6 to 20 μm, and rotifers were not able to ingest beads 12 μm in diameter or greater. Microparticulate diets and supplements for enrichment of this rotifer strain should have a particle size of 10 μm or less, with an optimal size of 4.5 μm. Development of effective feeds for enriching rotifers will likely result in higher survival and healthier fish larvae. The optimal enrichment period was 35 min, with a total ingested bead volume of 18 pl pL per rotifer, corresponding to approximately 1.18% of the volume of the rotifer. Furthermore, the influence of different water temperatures on gut passage time was determined and we found that exposure to 4 °C resulted in cessation of bead defecation after an initial loss of 32% in the first hour. We did not study the effects of cold water on reducing swimming activity or survival of rotifers that could negatively affect their availability as prey for fish larvae. These results will be useful for optimizing methods to enrich rotifers with artificial nutrients in order to improve their nutritional value as a live feed for rearing fish larvae. References Bern, L., 1990. Size related discrimination of nutritive and inert particles by freshwater zooplankton. J. Plankton. Res. 12, 1059–1067. Chotiyaputta, C., Hirayama, K., 1978. Food Selectivity of the rotifer Brachionus plicatilis feeding on phytoplankton. Mar. Biol. 45, 105–111. Ciros-Perez, J., Gomez, A., Serra, M., 2001. On the taxonomy of three sympatric sibling species of the Brachionus plicatilis (Rotifera) complex from Spain, with the description of B-ibericus n. sp. J. Plankton. Res. 23, 1311–1328.

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Cossins, A.R., Bowler, K., 1987. Temperature Biology of Animals. Chapman and Hall, New York, p. 339. DeMott, W.R., 1986. The role of taste in food selection by freshwater zooplankton. Oecologia 69, 334–340. Dhert, P., Rombaut, G., Suantika, G., Sorgeloos, P., 2001. Advancement of rotifer culture and manipulation techniques in Europe. Aquaculture 200 (1–2), 129–146. Dhert, P., 1996. Rotifers. In: Soreloos, P., Lavens, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture. Fisheries Technical Paper no. 361. Food andAgriculture Organization of the United Nations, Rome, pp. 49–78. Fontaneto, D., Melone, G., 2006. Postembryonic development of hard jaws (trophi) in a species belonging to the Brachionus plicatilis complex (Rotifera, Monogononta): a morphometric analysis. Microsc. Res. Tech. 69, 296–301. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech. 3, 294–299. Gomez, A., Serra, M., Carvalho, G.R., Hunt, D.H., 2002. Speciation in ancient cryptic species complexes: evidence from the molecular phyologeny of Brachinous plicatilis (Rotifera). Evolution 56 (7), 1431–1444. Guindon, S., Gasquel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Hall, B., 2004. Update #1: Better gap penalties, In: Hall, B. (Ed.), Basic Elements in Creating and Presenting Trees. In Phylogenetic Trees Made Easy, Second edition. Sinauer, Sunderland, MA, pp. 66–88. Hamre, K., 2006. Nutrition in cod (Gadus morhua) larvae and juveniles. ICES J. Mar. Sci. 63, 267–274. Hansen, B., Wernberg-Møller, T., Wittrup, L., 1997. Particle grazing efficiency and specific groth efficiency of the rotifer Brachionus plicatilis (Muller). J. Exp. Mar. Biol. Ecol. 215, 217–233. Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R., 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B. Biol. Sci. 270, 313–322. Heerkloss, R., Hlawa, S., 1995. Feeding biology of two brachionid rotifers: Brachionus quadridentatus and Brachionus plicatilis. Hydrobiologia 313/314, 219–221. Helfman, G., Colette, B., Facey, D., 1997. The Diversity of Fishes. Blackwell Science, Massachusetts, p. 512. Hino, A., Hirano, R., 1980. Relationship between body size of the rotifer Brachionus plicatilis and the maximum size of particles ingested. Bull. Jpn. Sot. Sci. Fish 46, 1217–1222. Hlawa, S., Heerkloss, R., 1994. Experimental studies into the feeding biology of rotifers in brackish water. J. Plankton Res. 16 (8), 1021–1038. Hotos, G.N., 2003. Growth, filtration and ingestion rate of the rotifer Brachionus plicatilis fed with large (Asteromonas gracilis) and small (Chlorella sp.) celled algal species. Aquac. Res. 34, 793–802. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Jacobs, J., 1974. Quantitative measurement of food selection — a modification of the forage ratio and Ivlev's selectivity index. Oecologia 14, 413–417. Keane, T.M., Creevey, C.J., Pentony, M.M., Naughton, T.J., McInerney, J.O., 2006. Assessment of methods for amino acid matrix selection and their use on empirical

data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol. Biol. 6, 29. Kleinow, W., Wratil, H., Kuhle, K., Esch., B., 1991. Electron microscope studies of the digestive tract of Brachionus plicatilis (Rotifera). Zoomorphology 111, 67–80. Kolkovski, S., 2001. Digestive enzymes in fish larvae and juveniles — implications and applications to formulated diets. Aquaculture 200 (1–2), 181–201. Kumlu, M., Jones, D.A., 1995. The effect of live and artificial diets on growth, survival, and trypsin activity in larvae of Penaeus indicus. J. World Aquac. Soc. 26 (4), 406–415. Lubzens, E., Tandler, A., minkoff, G., 1989. Rotifers as food in aquaculture. Hydrobiologia 186/187, 387–400. Mills, S., Lunt, D.H., Gomez, A., 2007. Global isolation by distance despite strong regional phylogeography in a small metazoan. MBC Evol. Biol. 7 (1), 225. Montagnes, D.J.S., Klimmance, S.A., Tsounis, G., Gumbs, J.C., 2001. Combined effect of temperature and food concentration on grazing rate of the rotifer Brachionus plicatilis. Mar. Biol. 139, 975–979. Önal, U., Langdon, C., 2000. Characterization of two microparticle types for delivery of food to altricial fish larvae. Aquac. Nutr. 6, 159–170. Papakostas, S., Dooms, S., Triantafyllidis, A., Deloof, D., Kappas, I., Dierckens, K., De Wolf, T., Bossier, P., Vadstein, O., Kui, S., Sorgeloos, P., Abatzopoulos, T.J., 2006a. Evaluation of DNA methodologies in identifying Brachionus species used in European hatcheries. Aquaculture 225, 557–564. Papakostas, S., Dooms, S., Christodoulou, M., Triantafyllidis, A., Kappas, I., Dierckens, K., Bossier, P., Sorgeloos, P., Abatzopoulos, T.J., 2006b. Identification of cultured Brachionus rotifers based on RFLP and SSCP screening. Mar. Biotechn. 8, 547–559. Ronneberger, D., 1998. Uptake of latex beads as size-model of planctonic rotifers. Hydrobiologoa 388, 445–449. Rothhaupt, K., 1990. Differences in particle size-dependent feeding efficiencies of closely related rotifer species. Limnol. Oceanogr. 35 (1), 16–23. Srivastsava, A., Hamre, K., Stoss, J., Chakrabarti, R., Tonheim, S.K., 2006. Protein content and amino acid composition of the live feed rotifer (Brachionus plicatilis): with emphasis on the water soluble fraction. Aquaculture 254, 534–543. Suatoni, E., Vicario, S., Rice, S., Snell, T., Caccone, A., 2006. An analysis of species boundaries and biogeographic patterns in a cryptic species complex: the rotifer Brachionus plicatilis. Mol. Phylogenet. Evol. 41, 86–98. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25 (24), 4876–4882. Tucker Jr., J.W., 1998. Marine Fish Culture. Kluwer Academic Publishers, Boston, p. 750. Vadstein, O., Gunvor, Ø., Yngvar, O., 1993. Particle size dependent feeding by the rotifer Brachionus plicatilis. Hydrobiologia 255/256, 261–267. Walford, J., Lam, T.J., 1987. Effect of feeding with microcapsules on the content of essential fatty acids in the live foods for the larvae of marine fishes. Aquaculture 61, 219–230. Watanabe, T., 1993. Importance of docosahexaenoic acid in marine larval fish. J. World Aquac. Soc. 24, 151–161. Yúfera, M., 2001. Studies on Brachionus (Rotifera): an example of interaction between fundamental and applied research. Hydrobiologia 446/447, 383–392. Yúfera, M., Fernandez-Diaz, C., Pascual, E., 1995. Feeding rates of gilthead seabream, Sparus aurata L., larvae on microcapsules. Aquaculture 134, 257–268.